Therapy-enhancing glucan

ABSTRACT

This invention provides a composition comprising an effective amount of (1-3),(1-4)-β-glucan derived from barley capable of enhancing efficacy of antibodies. This invention further provides the above compositions and a pharmaceutically acceptable carrier. This invention also provides a method for treating a subject with cancer comprising administrating the above-described composition to the subject.

This application is continuation of U.S. Ser. No. 10/621,027, filed Jul.16, 2003, which is a continuation-in-part of International ApplicationNo. PCT/US02/01276, Filed 15 Jan. 2002, claiming the benefit of U.S.Ser. No. 60/261,911, filed on 16 Jan. 2001, and the contents of thepreceding applications are incorporated by reference here into thisapplication in their entirety.

Throughout this application, various references are cited. Disclosuresof these publications in their entireties are hereby incorporated byreference into this application to more fully describe the state of theart to which this invention pertains.

BACKGROUND OF THE INVENTION

Monoclonal antibodies (MoAb) selective for tumors have therapeuticpotential.^(1,2) The introduction of hybridoma technology by Kohler andMilstein in 1975³ and the advances in molecular biologic techniques havegreatly expanded the potential of MoAb in human cancers. Anti-CEAantibody in colorectal cancer,⁴ anti-CD20 antibodies in lymphoma,⁵anti-HER2 antibodies in breast cancer,⁶ anti-tenascin antibodies inglial brain tumors,⁷ MoAb M195 against CD33 in acute leukemia⁸ andanti-TAG-72 antibodies in colon cancer⁹ have shown efficacy in clinicaltrials. Our laboratory has developed the MoAb 3F8 which targets theganglioside GD2 overexpressed on neuroblastoma. 3F8 has been shown tohave a high specificity and sensitivity in the radioimmunodetection ofminimal residual disease (MRD) in patients with NB,¹⁰ and a significantimpact when used as adjuvant therapy.¹¹

The immune basis of clinical tumor response to MoAb is at least twofold, direct cytotoxicity and induced immunity. Antibody dependentcell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity(CMC) are responsible for the direct killing of tumor cells. On theother hand, through tumor opsonization¹² or idiotype network,¹³tumor-specific immunity is induced. With this paradigm, how the bodyeliminates microbial pathogens remains highly relevant in our strategicapproach to cancer therapy. Since the first description of innateimmunity and acquired immunity model, several components have emergedcenter stage.¹⁴ Antibodies, complement, phagocytes, and “danger”receptors are core elements of innate immunity while antigen-presentingcells, T and B lymphocytes constitute essential players in acquiredimmunity. Despite the availability of tumor-selective monoclonalantibodies and the ample supply of phagocytes/natural killers, shrinkageof established tumors following antibody treatment alone, and theacquisition of specific immunity, are not common in both preclinicalmodels and cancer patients. The absence of a danger signal and thediminution of complement action by complement resistance proteins ontumor cells may explain the inefficiency of antibody mediated clinicalresponses.¹⁵ LPS and beta-glucan, being cell wall components of bacteriaand fungus, respectively, are potent danger signals to the immunesystems in all life-forms, from Drosophila to man.¹⁶ While LPS is tootoxic for human use, β-glucan is a relatively benign structuralcomponent extractable from cereals, mushrooms, seaweed and yeasts.¹⁷They are made up of 1,3-β-D-glucopyranosyl residues along which arerandomly dispersed single β-D-glucopyranosyl units attached by1,6-linkages, giving a comb-like structure. The 1,3-β-backbone and the1,6-linked branches were thought to be important for their immuneeffects. Lentinan (from Lentinus edodes, Basidiomycete family) with 1,6branches at mean of 3 main chain units, is licensed Japan for cancertreatment. Schizophyllan (from Schizophyllum commune, Basidiomycetefamily) and β-glucan from Baker's yeast (Saccharomyces cerevisiae) havealso similar structures. From seaweed, Laminarin (1,3 β-D-glucan with1,6-β side chain branching on every 10 glucose subunit along the polymerbackbone) has been extracted. Because of its smaller size and watersolubility, it was thought to have potential biologic utility. On theother hand β-glucan from barley, oat or wheat have mixed 1,3-β and1,4-β-linkage in the backbone, but no 1,6-β branches, and generallyhigher molecular weights and viscosities. In addition, they have not yetbeen tested for their in vivo immunomodulatory effects in cancer models.

This invention discloses that oral beta-glucans derived from barley oroats can greatly enhance the anti-tumor activity of anti-tumormonoclonal antibodies in xenograft models. Given the low toxicity oforal β-glucan, their role in cancer therapy deserves careful study.

SUMMARY OF THE INVENTION

This invention provides a composition comprising an effective amount ofglucan capable of enhancing efficacy of antibodies. In an embodiment,the antibody is a monoclonal antibody. In a further embodiment, theantibody is an antibody against cancer.

The cancer is recognized by antibodies, and which includes but notlimited to neuroblastoma, melanoma, non-Hodgkin's lymphoma, Epstein-Barrrelated lymphoma, Hodgkin's lymphoma, retinoblastoma, small cell lungcancer, brain tumors, leukemia, epidermoid carcinoma, prostate cancer,renal cell carcinoma, transitional cell carcinoma, breast cancer,ovarian cancer, lung cancer colon cancer, liver cancer, stomach cancer,and other gastrointestinal cancers.

This invention further provides the above compositions and apharmaceutically acceptable carrier, thereby forming pharmaceuticalcompositions.

This invention also provides a method for treating a subject with cancercomprising administrating the above-described composition to thesubject.

This invention provides a composition comprising effective amount ofglucan capable of enhancing efficacy of vaccines. In an embodiment, thevaccine is against cancer. This invention also provides the abovecompositions and a pharmaceutically acceptable carrier, thereby forminga pharmaceutical composition.

This invention also provides a method of treating a subject comprisingadministrating the above pharmaceutical composition to the subject. Inan embodiment, the subject is a human subject. In an embodiment, thevaccine is against infectious agents. The infectious agents include butare not limited to bacteria, viruses, fungi, or parasites.

This invention provides a composition comprising effective amount ofglucan capable of enhancing efficacy of natural antibodies. In anembodiment, the antibodies are against cancer. In another embodiment,the antibodies are against infectious agents. The infectious agentsinclude but are not limited to bacteria, viruses, fungi, or parasites.

This invention provides a composition comprising effective amount ofglucan capable of enhancing host immunity. In another embodiment, theimmunity is against cancer or infectious agents.

This invention also provides a composition comprising effective amountof glucan capable of enhancing the action of an agent in preventingtissue rejection. In another embodiment, the tissue is transplantedtissue or transplanted organ. In another embodiment, the tissue is thehost as in graft-versus-host reactions.

This invention also provides the above compositions, wherein the glucanare. 1,3-1,4 mixed linkage, without 1,6 branches.

The invention further provides the above compositions, wherein theglucan is of high molecular weight. In an embodiment, the molecularweight of the glucan ranges from 250,000 to 450,000 daltons. Thisinvention provides the above compositions, wherein the glucan is derivedfrom barley, oat, wheat or moss.

This invention provides the above compositions, wherein the glucan isstable to heat treatment. In an embodiment, the composition is stableafter boiling for 3 hours.

This invention provides the above compositions, wherein oral route isadopted when taken into a subject. In an embodiment, the effective doseis about >=25 mg/kg/day, five days a week for a total of 2-4 weeks.

DETAILED DESCRIPTION OF THE FIGURES

First Series of Experiments

FIG. 1. Synergistic effect of MoAb and β-glucan in LAN-1 Two millionLAN-1 neuroblastoma cells were xenografted subcutaneously in Balb/cathymic mice. Treatment started in groups of 5 mice each, 2 weeks aftertumor implantation when visible tumors reached 0.7-0.8 cm diameter. 3F8group (solid circles) was treated with 200 ug of intravenous 3F8injected through the retroorbital plexus twice weekly (M and 3F8+BGgroup (open circle) was treated with 200-ug i.v. 3F8 twice weekly plusoral Barley β-glucan (BG medium viscosity) 400 ug daily by gavage for atotal of 21 days. BG group (open triangle) received β-glucan alone, 400ug po daily for 21 days. Tumor size was measured from the first day oftreatment, and the product of the largest diameters expressed as percentof that on day 0 of treatment. While BG alone and 3F8 alone showed noanti-tumor effect, the BG+3F8 group showed highly significant tumorshrinkage and suppression (p<0.001).

FIG. 2. Synergistic effect of MoAb and β-glucan in NMB-7 Experiment inFIG. 1 was repeated with the neuroblastoma cell line NMB7, a slowergrowing line. Again BG alone (open triangle) and 3F8 alone (solidcircles) showed no anti-tumor effect, the BG+3F8 group (open circle)showed highly significant tumor shrinkage and suppression (p<0.001).Y-axis is relative tumor size in percent and X-axis the number of daysfrom first treatment.

FIG. 3. Dose response of intraperitoneal (ip) β-glucan Two million NMB-7xenografted nude mice were treated at the time of visible tumors with3F8 alone, normal saline control, or 3F8 plus increasing doses ofintraperitoneal BG (4 ug [solid diamond], 40 ug [open square], 400 ug[large open circle]) or 400 ug of po BG [small open circle], or 400 ugof ip Lentinan [open diamond]. Highly significant tumor shrinkage andsuppression was shown in the combination groups except at 4 ug of BGdose. Oral BG appeared to be more effective than ip BC.

FIG. 4. Dose response of oral β-glucan NMB-7 xenografted in nude micewere treated as in FIG. 3 except that dose response of oral β-glucan (4ug [open diamond], 40 ug [open triangle], 400 ug [open circle]) wascompared to 400 ug of ip BG [solid square]. Control group receivedsaline [solid circle]. 400 ug po was again highly significant ineradicating or suppressing tumor growth. 400 ip appeared to be aseffective as 40 ug po. 4 ug was the least effective.

FIG. 5. Dose response of oral β-glucan in LAN-1 Five million LAN-1 cellswere planted subcutaneously. Tumor growth was more rapid compared to 2million NMB-7 cells. Again 4 ug [solid squares], 40 ug [solid triangle]were no different from controls. Only 400 ug po [open circle] and 4000ug Po [open square] showed significant tumor eradication or suppression.

FIG. 6. Comparison of various β-glucans β-glucan [400 ug po qd] derivedfrom barley [7 days/wk open circle, M-F/week open triangle], Maitakemushrooms [solid triangles), and laminarin (open squares] were comparedin their synergism with antibody 3F8 against NMB-7 subcutaneousxenografts.

FIG. 7. More comparison of various β-glucans β-glucans (400 ug po qd)from different barley lots [large open circle, small open circle],lentinan [open diamond], PSK [cross] were compared to mannan [solidsquare], 3F8 only [open triangle] or no treatment [solid circle]. OnlyBG from barley showed syngergistic anti-tumor effect with antibody 3F8against LAN-1 xenografts.

FIG. 8. D-fraction Maitake Mushroom β-glucan [open square] had noanti-tumor effect when compared to barley β-glucan alone [solid circle],3F8 alone [open triangle], in contrast to barley β-glucan plus 3F8 [opencircle] which was highly effective.

FIG. 9. Barley β-glucan of large molecular weight is more effectiveβ-glucan of 40K [solid square], 123K [open triangle], 183K [opensquare], 254K [open diamond], and 359K [open circle] were tested at 40ug po daily dose in combination with 3F8 against LAN-1 subcutaneousxenografts. The larger the size of the β-glucan, the more effective thesynergistic effect.

FIG. 10. β-glucans of low molecular weight and low viscosity wasineffective β-glucans of various viscosities [40 ug po qd] derived frombarley and oats were tested in combination with 3F8 against LAN-1subcutaneous xenografts. Barley medium viscosity [large open circle],barley high viscosity [open square], oat medium viscosity [small opencircle, dotted line], and oat high viscosity [open square, dotted line]were all effective in shrinking and suppressing tumor growth, incontrast to low viscosity barley β-glucan [solid square].

FIG. 11. Removal of NK cells by anti-Asialo GM1 antiserum in LAN-1xenograft decreased but did not eliminate the anti-tumor effect ofbarley β-glucan plus 3F8.

FIG. 12. Removal of NK cells by anti-Asialo GM1 antiserum in NMB-7xenograft again decreased but did not eliminate the anti-tumor effect ofbarley β-glucan plus 3F8.

FIG. 13. 3F8-F(ab′)2 fragment [solid square], nonspecific human IgG[small solid square] or IgM [solid diamond] have no anti-tumor effectwhile 3G6 (IgM anti-GD2, open circle) was almost as effective as 3F8(IgG3 anti-GD2, open triangle).

FIG. 14. Barley β-glucan synergizes with R24 anti-GD3 antibody inSKMel28 melanoma xenografts in nude mice In contrast to β-glucan control[solid diamond], and R24 control [solid circle], the combination of R24and β-glucan [open circle] significantly suppressed tumor growth.

FIG. 15. Barley β-glucan synergizes with 3F8 anti-GD2 antibody againstB16D14 murine melanoma in C57Bl/6 mice In contrast to saline control[solid circle], β-glucan control [solid triangle], and 3F8 control[solid square], the combination of 3F8 and β-glucan [open circle]significantly suppressed tumor growth.

FIG. 16. Barley β-glucan synergizes with 3F8 anti-GD2 antibody againstB16D14-KbKd murine melanoma in C57Bl/6 mice In contrast to 3F8 control[solid circle], the combination of 3F8 and β-glucan [open circle]significantly suppressed tumor growth.

FIG. 17. Barley β-glucan plus 3F8 did not affect GD2-negative B16melanoma in C57Bl/6 mice The combination of 3F8 and β-glucan [solidcircle] did not significantly suppress tumor growth when compared tocontrols [open circle].

FIG. 18. Barley β-glucan synergizes with 3F8 anti-GD2 antibody againstEl4 murine lymphoma in C57Bl/6 mice In contrast to control [solidcircle], the combination of 3F8 and β-glucan [open circle] significantlysuppressed tumor growth.

FIG. 19. BARLEY Glucan syngergizes with 3F8 in prolonging survival fromNMB7 neuroblastoma. A established neuroblastoma NMB7 xenografts treatedwith 3F8 and barley β-glucan (open circles) had significantly longermedian survival >300 days compared to 30 days in the control mice (solidtriangle) treated with saline alone, 3F8 alone, or β-glucan alone(p<0.001). Long-term survival was 56% in the treatment group and 5% inthe control group.

FIG. 20. BARLEY glucan synergizes with 3F8 in prolonging survival fromLAN-1 neuroblastoma. In nude mice bearing established LAN1 xenograftsmedian survival increased from 20 days in the control group (n=38, solidtriangles) to 42 days in the 3F8 plus glucan group (n=48, open circles,p<0.001).

Second Series of Experiments

FIG. 21. Synergistic effect of MoAb and β-glucan in neuroblastomaxenografts. Two million neuroblastoma cells (21A: LAN-1, 21B: NMB7,21C:SK-N-ER) were xenografted subcutaneously in athymic Balb/c mice.Treatment started in groups of 5 mice each, 2 weeks after tumorimplantation when visible tumors reached 0.7-0.8 cm diameter. 3F8 group(solid circles) was treated with 200 ug of intravenous 3F8 injectedthrough the retroorbital plexus twice weekly (M and Th). 3F8+BG group(open circle) was treated with 200 ugi.v.3F8 twice weekly plus oralβ-glucan (BG) 400 ug daily by gavage for a total of 21-29 days. BG group(open triangle) received 400 ug oral β-glucan alone. Tumor size wasmeasured from the first day of treatment, and the product of the largestdiameters expressed as percent of the size on day 0 of treatment.Vertical bars represent standard errors, which were similar for theglucan and the 3F8 alone groups. While BG alone and 3F8 alone showed noanti-tumor effect, the BG+3F8 group showed highly significant tumorshrinkage and suppression (p<0.01).

FIG. 22. Dose response of intraperitoneal (ip) β-glucan. Two millionNMB7 xenografted athymic nude mice were treated at the time of visibletumors with 3F8 alone, normal saline control, or 3F8 plus increasingdoses of intraperitoneal BG (4 ug [solid diamond], 40 ug [open square],400 ug [large open circle]) or 400 ug of po BG [small open circle].Highly significant tumor shrinkage and suppression was shown in thecombination groups except at 4 ug of BG dose. Oral BG appeared to bemore effective than ip BG.

FIG. 23. Dose response of oral β-glucan. NMB7 xenografted in nude micewere treated as in FIG. 23 except that dose response of oral β-glucan (4ug [open diamond], 40 ug [open triangle], 400 ug [open circle]) wascompared to 400 ug of ip BG [solid square]. Control group receivedsaline [solid circle]. 400 ug po was again highly significant ineradicating or suppressing tumor growth. 400 ip appeared to be aseffective as 40 ug po. 4 ug was the least effective.

FIG. 24. Removal of NK cells by anti-Asialo GM1 antiserum on β-glucaneffect in LAN-1 xenografts decreased but did not eliminate theanti-tumor effect of β-glucan plus 3F8.

FIG. 25. Barley β-glucan syngergized with 3F8 in prolonging survival,from NMB7 neuroblastoma. A (n=22) with established neuroblastoma NMB7xenografts treated with 3F8 and barley β-glucan (solid line) hadsignificantly longer median survival (median 166 days) compared tocontrol mice (n=34, broken line, median 30 days) treated with salinealone (n=10), 3F8 alone (n=8), or β-glucan alone (n=16) (p<0.001).Long-term survival was 47% in the treatment group and 3% in the controlgroup.

FIG. 26. Barley β-glucan synergized with 3F8 in prolonging survival fromLAN-1 neuroblastoma. In nude mice bearing established LAN-1 xenografts,median survival increased from 21 days in the control group (n=55,broken line) to 54 days in the 3F8 plus glucan group (n=82, solid line,p<0.001).

Third Series of Experiments

FIG. 27. Oral β-glucan synqergizes with 3F8 in prolonging survival fromneuroblastoma. with established NMB7 xenografts (0.7-0.8 cm diametertumor at the beginning of treatment) were treated with 3F8 (200 ug twicea week iv) and 400 ug of β-glucan po daily for a total of 3 weeks.Control mice received either saline alone (n=10, broken line), 3F8 alone(n=8, dashed line), or β-glucan (n=16, dotted line) alone. Mediansurvival was 30 days in control groups and 166 days in the treatment(3F8 plus β-glucan, n=22) group (p<0.001). Ten (45%) in the combinationgroup survived long term with a median follow-up of 248 days. Only onemouse in any of the control groups (<5%) remained alive during theexperiment.

FIG. 28. Synergy of Oral barley β-glucan with (A) R24 (anti-GD3)antibody against SKMe128 melanoma xenografts in nude mice. In contrastto β-glucan control [solid diamonds], and R24 control [solid circles],the combination of R24 and β-glucan [open circles] significantlysuppressed tumor growth (tumor growth rate reduced for combinationtreatment by 1.2%, 95% CI −0.1%, 2.5%, p=0.06) (B) 528 (anti-EGF-R) MoAbagainst epidermoid carcinoma A431 xenografts in nude mice. In contrastto β-glucan+455 (IgG1 noncomplement fixing) control [solid sqaures], and528 MoAb alone [solid circles], the combination of 528 MoAb and β-glucan[open circles] significantly suppressed tumor growth (tumor growth ratereduced for combination treatment by 1.4%, 95% CI −0.7%, 3.5%, p=0.17).(C) Herceptin (anti-HER2) antibody against human breast carcinoma BT474xenografts in nude mice. In contrast to control [n=4, solid circles],Herceptin [n=9, open squares], or β-glucan control [n=7, solid squares],the combination of Herceptin and β-glucan [n=12, open circles]significantly suppressed tumor growth (tumor growth rate reduced forcombination treatment by 1.9%, 95% CI 0.7%, 3%, p=0.002).

Fourth Series of Experiments

FIG. 29A. Baseline MIBG scan of patient 5. Extensive osseous metastasiscan be seen in the femora, fibulae, pelvis, rib, left scapula, rightclavicle, humeri, skull and spine. Heart, liver, stomach and colonuptakes are physiologic.

FIG. 29B. MIBG Scan of same patient 2 months later, following a singlecycle of therapy. Areas of metastases have significantly improved.

Fifth Series of the Experiment

FIG. 30. Subcutaneous xenograft growth in SCID mice. SCID mice withestablished subcutaneous Daudi (n=9) (Fig 30A), Hs445 (n=5)(Fig. 30B),EBV-derived LCL (n=9) (Fig 30C) and RPMI 6666 (n=10; data not shown)xenografts were treated either with 200 ug intravenous rituximab twiceweekly for 8 doses (▪), 400 ug (1→3), (1→4)-D-β-glucan administeredorally via intragastric gavage daily for 29 days (Δ) or a combination ofrituximab and (1→3), (1→4)-D-β-glucan (x), or left untreated (♦).Percentage tumor growth is plotted on y-axis and days after treatmentwas commenced on x-axis. Error bars represent SEM and have been shownonly for rituximab alone and combination groups. For all xenografts,only combination treatment was associated with reduction in tumorgrowth. The reduction in tumor growth per day in the group receivingβ-glucan in addition to rituximab compared to rituximab alone was 2.0%(95% CI 1.3-2.7%; p<0.0005) for Daudi, 0.8% for EBV-derived LCL (95% CI0.4-1.2%; p<=001), 2.2% for Hs445 (95% C.I. 1.2%-3.2%; p=0.0009), and1.8% for RPMI6666 (95% CI 1.0-2.7%; p<0.0002; data not shown)xenografts.

FIG. 31. Survival in SCID mice with disseminated lymphoma xenografts.5×10⁶ Daudi (FIG. 2A) or Hs445 (FIG. 2B) cells in 100 μl normal salinewere injected intravenously (IV) into SCID mice. Mice were treatedeither with 200 ug intravenous rituximab twice weekly for 8 doses( - - - ), 400 ug (1→3), (1→4)-D-β-glucan administered orally viaintragastric gavage daily for 29 days ( . . . ) or a combination ofrituximab and (1→3), (1→4)-D-β-glucan (

), or left untreated (

) commencing 10 days after tumor implantation. Tumors grew systemicallyand mice became paralyzed when tumor cells infiltrated the spinal canal,resulting in hind-leg paralysis. Mice were sacrificed at onset ofparalysis or when animals lost 10% of their body weight. Kaplan-Maiersurvival curves for the various groups are shown in FIGS. 2A (Daudi) and2B (Hs445). Mice treated with a combination of (1→3), (1→4)-D-β-glucanand rituximab had a significantly increased survival when compared toall other treatment groups (p<0.0005 for Daudi and p=0.001 for Hs445) orwhen compared to rituximab alone (p<0.0005 for Daudi and p=0.01 forHs445). Median survival for mice with no treatment, rituximab alone, BG,and rituximab+BG groups was 27,71,43 and 124 days respectively for Daudixenografts, and 12, 16, 31 and 243 days respectively for Hs445xenografts.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a composition comprising an effective amount ofglucan capable of enhancing efficacy of antibodies.

In an embodiment, the antibody is a monoclonal antibody. In a furtherembodiment, the antibody is an antibody against cancer. In anotherembodiment, the antibody is a tumor-binding antibody. In a furtherembodiment, the antibody is capable of activating complement. In a stillfurther embodiment, the antibody is further capable of activating theantibody dependent cell-mediated cytotoxicity.

In an embodiment, the antibody is directed at the epidermal growthfactor receptor. In a further embodiment, the antibody is 528 or C225.

In another embodiment, the antibody is directed to a ganglioside. In afurther embodiment, the ganglioside is GD3. In a still furtherembodiment, the antibody is R24.

In a separate embodiment, the ganglioside is GD2. In a furtherembodiment, the antibody is 3F8.

In an embodiment, the antigen recognized by the antibody is CD20. In afurther embodiment, the antibody is Rituximab.

In another embodiment, the antigen is CD25. In a further embodiment, theantibody is Dacluzimab.

In a separate embodiment, the antigen is Her2/neu. In a furtherembodiment, the antibody is Herceptin.

In another embodiment, the antigen is CD22. In a further embodiment, theantibody is Epratuzumab.

The cancer is recognized by antibodies, and it includes but is notlimited to neuroblastoma, melanoma, non-Hodgkin's lymphoma, Epstein-Barrrelated lymphoma, Hodgkin's lymphoma, retinoblastoma, small cell lungcancer, brain tumors, leukemia, epidermoid carcinoma,⁴⁰ prostatecancer,^(40,41) renal cell carcinoma,⁴⁰ transitional cell carcinoma,⁴⁰breast cancer,^(42,43) ovarian cancer,⁴⁰ lung cancer, colon cancer,⁴⁰liver cancer, stomach cancer, and other gastrointestinal cancers.

This invention further provides the above compositions and apharmaceutically acceptable carrier, thereby forming pharmaceuticalcompositions.

This invention also provides a pharmaceutical composition comprising acombination as described above and a pharmaceutically acceptablecarrier. For the purposes of this invention, “pharmaceuticallyacceptable carriers” means any of the standard pharmaceutical carriers.Examples of suitable carriers are well known in the art and may include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution and various wetting agents.Other carriers may include additives used in tablets, granules andcapsules, etc. Typically such carriers contain excipients such asstarch, milk, sugar, certain types of clay, gelatin, stearic acid orsalts thereof, magnesium or calcium stearate, talc, vegetable fats oroils, gum, glycols or other known excipients. Such carriers may alsoinclude flavor and color additives or other ingredients. Compositionscomprising such carriers are formulated by well-known conventionalmethods.

This invention also provides a method for treating a subject with cancercomprising administrating the above-described composition to thesubject.

This invention provides a composition comprising an effective amount ofglucan capable of enhancing efficacy of vaccines. In an embodiment, thevaccine is against cancer.

This invention also provides the above compositions and apharmaceutically acceptable carrier, thereby forming a pharmaceuticalcomposition.

This invention also provides a method of treating a subject comprisingadministrating the above pharmaceutical composition to the subject. Inan embodiment, the subject is a human subject.

In an embodiment, the vaccine is against infectious agents. Theinfectious agents include but are not limited to bacteria, viruses,fungi, or parasites.

This invention provides a composition comprising an effective amount ofglucan capable of enhancing efficacy of natural antibodies.

In an embodiment, the antibodies are against cancer.

In another embodiment, the antibodies are against infectious agents. Theinfectious agents include but are not limited to bacteria, viruses,fungi, or parasites.

This invention provides a composition comprising an effective amount ofglucan capable of enhancing host immunity. In another embodiment, theimmunity is against cancer or infectious agents.

This invention also provides a composition comprising an effectiveamount of glucan capable of enhancing the action of an agent inpreventing tissue rejection.

In an embodiment, the agent is an antibody. In a further embodiment, theantibody modulates T-cell function. In a still further embodiment, theantibody is anti-CD25 or anti-CD3.

In a separate embodiment, the antibody modulates B-cell function. Inanother embodiment, the antibody is anti-CD20.

In another embodiment, the tissue is transplanted tissue or transplantedorgan. In another embodiment, the tissue is the host as ingraft-versus-host reactions.

This invention also provides the above compositions, wherein the glucanare 1,3-1,4 mixed linkage, without 1,6 branches.

The invention further provides the above compositions, wherein theglucan is of high molecular weight. In an embodiment, the molecularweight of the glucan ranges from 250,000 to 450,000 daltons.

This invention provides the above compositions, wherein the glucan isderived from barley, oat, wheat, or moss.

This invention provides the above compositions, wherein the glucan isstable to heat treatment. In an embodiment, the composition is stableafter boiling for 3 hours.

This invention provides the above compositions, wherein the oral routeis adopted when administered a subject. In an embodiment, the effectivedose is about >=25 mg/kg/day, five days a week for a total of 2-4 weeks.

This invention provides a composition for oral uptake of substancecomprising an appropriate amount of carbohydrates. In an embodiment, thecarbohydrate is glucan.

When administered orally, glucan is taken up by macrophages andmonocytes which carry these carbohydrates to the marrow andreticuloendothelial system from where they are released, in anappropriately processed form, onto myeloid cells including neutrophils,and onto lymphoid cells including natural killer (NK) cells. Thisprocessed glucan binds to CR3 on these neutrophils and NK cells,activating them in tumor cytotoxicity in the presence of tumor-specificantibodies.

Since macrophage and monocytes ingest glucan (whether soluble, gel orparticle) from the gut, glucan is a potential conduit for gene therapy.Unlike proteins, DNA or plasmids are relatively heat-stable, and can beeasily incorporated into warm soluble barley glucan which gels whencooled to room or body temperature. When mice are fed these DNA-glucancomplexes, reporter genes can be detected in peripheral blood monocytesand macrophages within days. More importantly these reporter genes areexpressed in these cells, a few days after ingestion of these DNAcomplexes. These findings have potential biologic implications. Glucanand similar carbohydrates may be conduits for DNA or plasmids to getinto the human body. Oral glucan may be a convenient vehicle forcorrecting genetic defects of macrophages/monocytes, or administeringgenetic vaccines.

As it can easily be appreciated by an ordinary skilled artisan, othercarbohydrates capable of functioning like glucan could be identified andused in a similar fashion. One easy screening for such carbohydrates canbe established using glucan as the positive control.

The glucan includes but is not limited to 1,3-1,4 mixed linkage-glucan,and the glucan is of high molecular weight.

The substance which could be delivered orally includes but is notlimited to peptides, proteins, RNAs, DNAs, and plasmids. Other smallmolecules and compounds may be used as well.

This invention further provides a pharmaceutical composition comprisingan effective amount of the above composition and a pharmaceuticallyacceptable carrier.

This invention also provides a method for introducing substance intocells comprising contacting the above compositions with said cells. Onecan use reporter genes or other markers to assess the efficiency of thesaid introduction. Reporter genes or markers are well known in themolecular biology field. In addition, this invention provides a methodfor introducing substance into a subject comprising administering to thesubject an effective amount of the above compositions.

This invention provides a method for treating a subject comprisingadministering to the subject an effective amount of the abovecomposition. In an embodiement, the method further comprises thesubstance.

This invention provides a method for treating a subject with geneticdisorder comprising administering to the subject an effective amount ofthe above-described composition and a substance capable of correctingsaid genetic disorder. The substance includes but is not limited to apeptide, protein, RNA, DNA, plasmid and other small molecule andcompound.

The invention will be better understood by reference to the ExperimentalDetails which follow, but those skilled in the art will readilyappreciate that the specific experiments detailed are only illustrative,and are not meant to limit the invention as described herein, which isdefined by the claims which follow thereafter.

EXPERIMENTAL DETAILS

Materials and Methods

Cell lines Human neuroblastoma cell lines LA-N-1 was provided by Dr.Robert Seeger, Children's Hospital of Los Angeles, Los Angeles, Calif.and NMB7 by Dr. Shuen-Kuei Liao (McMaster University, Ontario, Canada).Neuroblastoma cell lines SKNHM, SKNHB, SKNJD, SKNLP, SKNER, SKNMM, SKNCHand SKNSH were derived from patients with metastatic disease treated atMemorial Sloan-Kettering Cancer Center (MSKCC), New York, N.Y. Othercells lines, Daudi, RMPI 6666, SKMel-28, A431, B16 were derived fromAmerican Type Culture Collection (ATCC), Rockville, Md. The cell linesB16, B16D14 were kindly provided by Dr. Kenneth Lloyd of MemorialSloan-Kettering Cancer Center. Kb transfected (B16D14 Kb) and (Kb+Kd)transfected (B16D14 KbKd) were kindly provided by Dr. Michel Sadelain,MSKCC. Cell lines were cultured in 10% defined calf serum (Hyclone,Logan, Utah) in RPMI with 2 mM L-glutamine, 100 U/ml of penicillin(Sigma, St. Louis, Mo.), 100 ug/ml of streptomycin (Sigma), 5% CO2 in a37° C. humidified incubator. Normal human mononuclear cells wereprepared from heparinized bone marrow samples by centrifugation across aFicoll-Hypaque density separation gradient.

Antibodies Monoclonal antibodies 3F8 (mouse IgG3) and 3G6 (mouse IgM),and 8H9 (mouse IgG1) reactive with neuroblastoma have been previouslydescribed. They were produced by as ascites and purified by affinitychromatography: protein A (Pharmacia, Piscataway, N.J.) for 3F8,¹⁸protein G (Pharmacia) for 8H9,¹⁹ and C1q-sepharose (Pierce Chemicals)for 3G6.^(18,20) These antibodies are >90% pure by SDS-PAGE. F(ab′)2fragments were prepared by pepsin digestion as previously reported.²¹Anti-GD3 antibody (R24) was kindly provided by Dr. Paul Chapman ofMSKCC.²² FLOPC21, an IgG3 myeloma, was purchased from Sigma Chemicals,St. Louis, Mich. TIB114 (N.S.7) a hybridoma secreting an IgG3 controlantibody was obtained from ATCC. Rabbit anti-asialo-GM1 antibody (WakoPure Chemical Industries, Ltd, Osaka, Japan) diluted to 1 mg/ml ofprotein was administered at 200 ul ip on days 0, 1, 2, 7, 14, 21.Rituximab, anti-CD20 antibody was purchased from Genentech, Inc., CA.

Indirect immunofluorescence 1 million target cells were washed in PBSand then spun at 180×g for 5 min. The pellets were then reacted with 100μl of 15 μg/ml 8H9 at 4° C. for 1 hour. After washing the cells with PBSthey were allowed to react with 100 μl FITC-conjugated goat F (ab′)2anti-mouse IgG+IgM, (Biosource International, Camarillo, Calif.) at 4°C. ¹⁸ Flow cytometric analysis was performed using FACSCaliburImmunocytometer (Becton-Dickinson Immunocytometry Systems, San Jose,Calif.).

Glucan 1,3-1,4-β-glucan derived from barley, 1,3-1,6-β-glucan(Laminarin) from seaweed (Laminaria digitata), and mannan were purchasedfrom Sigma Co. 1,3-1,6-β-glucan (Lentinan) was obtained from DrugSynthesis and Chemistry Branch, Developmental Therapeutics Program,Division of Cancer Treatment, National Cancer Institute, Bethesda, Md.Maitake mushroom glucan (containing 1,3-1,6-β-glucan extracted fromGrifola frondosa) D-fraction was obtained from Maitake Products Inc.,Paramus, N.J. Barley and oats β-glucans of various molecular sizes(measured by high performance size-exclusion chromatography [HPSEC] withmultiple angle laser scattering [MALLS] detection) and viscosities(measured in cSt units) were obtained from Megazyme InternationalIreland Ltd., Bray, County, Ireland. Barley glucan was dissolved byboiling for 10 minutes in normal saline. A stock solution of Lentinan inDMSO (Sigma) was diluted in water before use.

Mice and treatment Athymic Balb/c and C57Bl/c mice were purchased fromJackson Laboratories, Bar Harbor, Me., and CB-17 SCID from Taconic.Tumor cells were planted (1-5×10(6) cells) in 100 ul of Madrigel (SigmaCo) subcutaneously. Following implantation, tumor sizes (maximum widthand lengths) were measured. Tumor size was calculated as product of the2 perpendicular diameters. Treatment studies started when tumor diameterreached 0.7 to 0.8 cm, usually by 14-21 days of tumor implantation. Micereceived antibody treatment intravenously (by retroorbital injection)twice weekly and glucan by gavage every day for a total 3-4 weeks (21-8days of glucan and 6-8 doses of antibody). Mice were weighed once a weekand tumor size measured twice a week. Mice were sacrificed when tumorsreached sizes that interfered with their well-being.

⁵¹Chromium (51Cr) release assay²³ In brief, 2×10³ of 51Cr-labeled targetcells were mixed with effector cells in a final volume of 0.2 ml ofmedium in 96-well flat-bottomed microtiter plates (Costar, Cambridge,Mass.). The plates were incubated for 4 h at 37° C. in 5% CO2 and thencentrifuged. 100 ul of assay supernatant was counted in a gamma counter.Target cell spontaneous chromium release ranged from 10 to 25%.

Results

Barley glucan synergizes with anti-GD2 antibody 3F8 in eradicating humanneuroblastoma. 3F8 is a murine IgG3 monoclonal antibody specific forganglioside GD2. It activates mouse and human complement, and mediateseffective ADCC against human neuroblastoma cells in vitro. Barley glucanwhen administered orally at 400 ug qd had no appreciable effect on tumorgrowth compared to antibody 3F8 given i.v. alone. However, when barleyglucan and 3F8 were used in combination, tumor growth was near totallysuppressed. In >40% of mice, NMB7 tumors remained permanently suppressedeven when treatment was stopped after 21 days. Similar observations weremade with neuroblastoma cell lines derived from different sources: NMB7,LAN-1 (FIGS. 1 and 2) and SK-N-ER (data not shown). Barley glucan wasequally effective when administered orally or intraperitoneally. Incontrast, for the GD2-negative rhabdomyosarcoma, HTB82, 3F8 plusβ-glucan treatment was ineffective (data not shown).

Dose response curve for ip barley glucan. When the dose ofintraperitoneal barley glucan was decreased 10-fold from 400 ug, it wasclear that 4 ug was no longer effective in synergizing with MoAb 3F8 insuppressing NMB7 growth. Interestingly, both ip lentinan and po glucan(at 400 ug po qd) were also effective (FIG. 3).

Oral barley glucan is as effective as ip glucan When oral barley glucanwas studied in NMB7 tumor (FIG. 4) followed after treatment for 80 days,similar dose response was found, i.e. while 400 ug oral regimen wascurative, breakthroughs were seen for the other dose levels, with 4 ugoral dose escaping sooner than 40 ug. Interestingly, 400 ug ip was onlyas effective as the 40 ug oral group, with late breakthrough tumorgrowths around the same time, unlike the 400 ug po group, where alltumors remained suppressed despite stopping all therapy after 21 days.Using the LAN1 tumor model, both 4 and 40 ug glucan were ineffectivecompared to 400 and 4000 ug of glucan per dose (FIG. 5). There was nosignificant body weight change in the treatment groups (after accountingfor tumor growth) irrespective of dose of glucan or combination withantibody 3F8. At necropsy on day 21, there were no appreciabledifference in the peripheral blood counts, cholesterol and bloodchemistry between mice receiving different glucan doses. There was alsono difference in the histologic appearances of organs in mice treatedwith glucan at any of the dose levels, when compared to control micethat received saline.

By the oral route, only certain glucans, and frequent dosing wereeffective. For NMB7 tumors (FIG. 6), 400 ug oral maitake was effectivein synergizing with antibody 3F8, although late breakthroughs were seen.A 5 day/week po barley glucan regimen was equally as effective as thedaily regimen. In contrast, a once a week or twice a week schedule ofbarley glucan was ineffective (data not shown). For the faster growingLAN1 tumors (FIG. 7) unlike barley glucan (lot #1 and lot #2), polentinan, PSK or mannan were all ineffective. The effect of Maitakeglucan was not significantly different from glucan dose or 3F8 alone(FIG. 8). Glucan from barley was more effective than that from oatdespite similarities in their molecular sizes (FIG. 11).

Barley glucans of large molecular weight is more effective In FIG. 9,barley glucans of different molecular sizes (40K, 123K, 183K, 254K,359K) were tested at an oral dose of 40 ug. Anti-tumor effect improvedwith increasing molecular weights, such that glucan of 359K size wasmost effective. Nevertheless, at high doses (e.g. 400 ug) even the lesseffective sizes, 40K and 128K showed some benefit (092900megazyme.xlsfrom folder megazyme). Glucan derived from oat also showed syngergisticanti-tumor effect when administered in the presence of 3F8. Bothmolecular size and viscosity appeared to be important for thisanti-tumor effect. For example barley glucan (327K, >100 cSt or 250K, 25cSt) and oat glucan (69 cSt or 20-30 cSt) were highly effective insynergizing with MoAb, whereas barley glucan of 137K and 5.6 cSt was not(FIG. 10).

Role of NK cells in glucan effect. Removal of NK cells using anti-AsialoGM1 antiserum eliminated a substantial amount, although not completelythe anti-tumor activity of glucan (FIGS. 11 and 12). Moreover in beigemice glucan was effective in synergizing with 3F8 (data not shown),suggesting that at least part of the anti-tumor activity was mediated byNK-independent cytotoxicity.

IgG3-F(ab′)2 or IgG1 antibodies do not have substantial anti-tumoractivity (FIG. 13) The role of Fc in mediating the anti-tumor effect ofglucan was apparent when Fc was removed by pepsin or when IgG1 isotype(data not shown) was used. Neither was able to activate complement ormediate efficient ADCC, and neither has significant anti-tumor effectwhen administered with 400 ug of oral glucan.

Barley Glucan synergizes with other complement fixing antibodies in awide spectrum of human tumors. IgG3 anti-GD3 antibody R24 synergizedwith po glucan in shrinking melanoma SKMel-28 xenografts (FIG. 14).Rituximab (humanized IgG1 anti-CD20) synergized with po glucan ineradicating EBV-lymphoma, Daudi lymphoma, and Hodgkin's disease.Although anti-EGF-R antibody 428 (mouse IgG2a) was able to suppressepidermal carcinoma⁴⁰ A431 tumor growth, 428 plus oral glucan was muchmore effective in eradicating tumors.

Cell line Antibody Subcutaneous tumor models Human xenografts NMB7 3F8LAN-1 3F8 SK-N-ER 3F8 SK-N- 3F8 SKMel-28 R24 EBV-lymphoma RituximabDaudi lymphoma Rituximab Hodgkin's disease Rituximab Epidermal Carcinoma528 Syngeneic tumors EL4 3F8 B16D14 3F8 B16D14-Kb 3F8 B16D14-Kb-Kd 3F8Metastatic tumor models Human xenografts Daudi 3F8 Syngeneic tumors EL43F8

Glucan synergizes with 3F8 in C57Bl/6 mice against syngeneic tumors.While our early experiments were focused on human xenografts in athymicor SCID mice, similar synergism was observed in immunologically intactmice grafted with GD2-positive B16 melanomas (B16D14 [FIG. 15], B16D14Kb, or B16D14 KbKd [FIG. 16]) or GD2-positive EL4 lymphoma [FIG. 18].Neither barley glucan nor 3F8 by itself showed anti-tumor effect. Incontrast the combination of glucan and 3F8 was able to suppress almostentirely tumor growth in C57Bl/6 mice. In control B16 tumors which wereGD2-negative, glucan plus 3F8 had no anti-tumor effect. We conclude thatthe glucan effect observed was not restricted to immune deficientanimals. In addition, it requires tumor-specific antibodies, i.e.effective only if tumor cells carried the target antigen.

Glucan synergizes with 3F8 in C57Bl/6 mice against metastatic tumors.When EL4 lymphoma cells were injected iv, mice developed widespreadtumors in their lungs, livers and at the site of injection and rapidlysuccumbed. Control animals were dead by 14 days following EL4 injection,while the group treated with i.v. 3F8 plus 400 ug glucan po (treatmentinitiated 5 days following EL4 injection) had significantly longersurvival.

Barley Glucan syngergizes with 3F8 in prolonging survival Nude mice(n=21) with established neuroblastoma NMB7 xenografts (0.7-0.8 cmdiameter tumor at the beginning of treatment) were treated with 3F8 (200ug twice a week iv) and 400 ug of barley β-glucan po daily for a totalof 3 weeks. Control mice (n=21) received either saline alone, 3F8 alone,or β-glucan alone. Median survival was 30 days in control and >300 daysin the treatment (3F8 plus β-glucan) group (p<0.001). Long-term survivalwas 56% in the treatment group and 5% in the control group (FIG. 19). Innude mice bearing established LAN1 xenografts (also 0.7-0.8 cm diametertumor at the beginning of treatment) median survival increased from 20days in the control group (n=38) to 42 days in the 3F8 plus glucan group(n=48) (p<0.001, FIG. 20).

Discussion

Using the human xenograft and syngeneic mouse tumor models, we have madethe following observations. Glucan derived from barley or oats cansynergize with monoclonal antibodies in suppressing or eradicatingtumors, while β-glucan or antibody alone has little anti-tumor effect.Anti-tumor response requires antibodies that activate complement,whether mouse IgM, mouse IgG3 or human IgG1. Glucans of high molecularweight 250K and viscosity (20 cSt) possess this special effect. Oralroute is at least equally (if not more) effective than theintraperitoneal route. It is a dose-dependent phenomenon, where 400 ugper dose is required for maximal effect. Natural killer cells are notessential for this glucan phenomenon, although they contribute to theanti-tumor effect. Normal T-cells and B-cells are not required for theanti-tumor effect since immune-deficient mouse strains demonstrate theglucan effect, whether athymic, SCID or SCID-beige mice are used. Inaddition, normal T-cells and B-cells do not interfere with this glucaneffect, as shown in the syngeneic C57Bl/6 mouse model. Most importantly,oral glucan is well-tolerated by all the mice tested so far, with nonoticeable change in body weight, blood counts or organ histologies,even at doses as high as 4 mg per dose per day.

Our findings differ significantly from previous observations andpredictions on the use of glucans in cancer treatment. In the past itwas thought that the 1,3-1,6-βlinkage was absolutely required for theglucan anti-tumor effect.¹⁷ This structure contains1,3-β-D-glucopyranosyl units along which are randomly dispersed singleβ-D-glucopyranosyl units attached by 1,6-linkages, giving a comb-likestructure (e.g. Lentinan, Schizophyllan, Laminarin, and glucan fromBaker's yeast). In these models, it was believed that T-cells cells wereactivated and indeed required for the anti-tumor effect. In addition, itwas believed that small molecular weight glucan should be more effectivethan high molecular weight glucan and that the most effectiveadministration should be intravenous or intraperitoneal routes. Indeed,Betafectin (PGG) was derived from a genetically engineered Saccharomycescerevisiae which makes 1,3-1,6-β-D glucans with weaker interchainassociations.²⁴ It was manufactured for i.v. injection to improvemacrophage function in the hope of reducing infectious complications andimproving wound healing. Barley glucan is a linear polymer with 1,3 and1,4 linkages; however, it is not a comb like structure. We did not findany anti-tumor effect of barley glucan when given alone. However, whenused in combinations with monoclonal antibodies, the syngergistic effectwas remarkable. In addition, glucans of high molecular weight and highviscosity appeared to be most effective, contrary to what one mightexpect for macromolecular transport. Although barley glucan activatesgranulocyte mediated ADCC in vitro (data not shown), the effects ofglucan may be indirect. It is not clear if the absorption of glucan isnecessary for its anti-tumor effect. The exact mechanism of how barleyglucan enhances the anti-tumor effect of monoclonal antibodies in vivois unknown.

One possible mechanism of action may relate to innate receptors forβ-glucan, in a hard-wired information network on phagocytes and lymphoidcells; receptors that normally recognize death signals and microbialmolecular patterns.²⁵ Monoclonal antibodies, either through Fcinteraction or through CR3 interaction with iC3b, direct cytotoxicity totumor cells, a process greatly enhanced by β-glucan activation ofeffector cells. This killing is immediate, nonclonal, and obligatory, aprocess often referred to as innate immunity. The consequence of thisinnate effector arm is the activation of costimulatory molecules andinduction of cytokines and chemokines that will enhance adaptiveimmunity to the tumor cells. Thus, activation of immunity is based upondiscrimination between dangerous and nondangerous antigens; and ifcancer can be viewed as constant danger to the immune system,^(26,27)memory T-cells will not become tolerized. β-glucan receptors belong to afamily of pattern recognition receptors (PRRs) specific forpathogen-associated molecular patterns (PAMPs). They are biosensors forinvading pathogens widely distributed in vertebrate and invertebrateanimals,²⁸ a nonclonal host defense pathway with structural andfunctional homologies in phylogenetic lineages that diverged over abillion years ago. A limited set of conserved signaling modules such asToll/IL-1R homology domain, the SIIK domain, the Rel homology domain andperhaps the leucine rich regions (LRR) domain, represent the originalbuilding blocks for PRRs. For example, insects respond to infection byrapid and transient synthesis of antimicrobial peptides by the fat bodyand hemocytes. In drosophila antibacterial peptides (cecropin, attacinand definsin) and anti-fungal peptide drosomycin are dependent on theToll pathway; this PRR activates a proteolytic cascade to act onSpatzle, or 18-Wheeler (18W) to form the active ligand for Toll.Activation of the human Toll homologue results in induction of IL-1,IL-6, IL-8, B7.1 and B7.2. With B7, signaling through CD28 occurs;T-cell become activated followed by expression of surface molecules suchas TNF-α and TNF-β, Fas ligand (L), CD40L, CD30L, and CD27L, as well assecretion of cytokines. Interestingly, for dendritic cells, when theyexpress B7, they stop antigen uptake (i.e. becoming nonendocytic) andassume their antigen-presenting role. Certain activation motifs such asLRR are present in Toll and the endotoxin receptor CD14; they are alsopresent intracellularly in plants, probably responsible for resistanceto intracellular pathogens.²⁸

Carbohydrate-rich antigens on bacteria or fungi can activate complement.Alternatively, specific antibodies can also deposit complementcomponents on pathogens or cancer cells, such as the C3b fragment of C3,which is rapidly proteolyzed into iC3b fragment by serum factor I. TheseiC3b fragments can glue pathogens or tumor cells to the iC3b-receptors(CR3, CD11b/CD18) on phagocytic cells and NK cells, stimulatingphagocytosis and/or cytotoxic degranulation. Thus, antibody andcomplement link innate and adaptive immunity by targeting antigens todifferent cells of the immune system, e.g. via CR3 and Fc for phagocyticcells, CR2 for B cells, and CR1, CR2, or CR3 for follicular dendriticcells.²⁹ For neutrophils, CR3-dependent phagocytosis requires ligationof two distinct binding sites, one for iC3b and a second site forβ-glucan. Without β-glucan, iC3b-opsonized target cells are resistant tokilling.³⁰ Microbes possess polysaccharides that can activate the lectindomain on CR3, leading to phagocytosis and cytotoxic degranulation. Incontrast, human cells (including tumors) lack these CR3-bindingpolysccharides, thus the inability of CR3 to mediate phagocytosis orextracellular cytotoxicity of tumor cells opsonized with iC3b. Thelectin site of CR3 can also influence transmembrane signaling ofendogenous neutrophil membrane GPI-anchored glycoproteins (CD14, CD16,CD59, CD87[uPAR]). In a mouse mammary tumor model, where there isnaturally occurring IgM and IgG antibodies, injection of yeast solubleβ-glucan could suppress tumor growth, an effect lost in C3 deficient orCD11b (CR3)-deficient mice.^(31,32) Since iC3b bound to a primaryprotein antigen can also enhance recognition and specific antibodysynthesis by antigen-specific B cells,³³ the presence of glucan pluscomplement activation may enhance B-cell response to pathogens or tumorcells.

If this syngergistic effect of β-glucan on antibodies is active inhumans, our findings may have broad clinical implications. First theefficacy of monoclonal antibodies in cancer (e.g. Herceptin, Rituximab,Dacluzimab, anti-GD2 and anti-EGF-R MoAb) can be potentially enhanced.³⁴Nevertheless, even though toxicity from glucan is expected to beminimal, the enhanced efficacy of MoAb may also increase MoAb-mediatedtoxicity. For example, the side effects of Herceptin on cardiacfunction, or anti-GD2 MoAb on neuropathic pain may be increased. Second,since the amount and quality of barley and oat glucan in daily foodintake can vary, future interpretations of efficacy trials using MoAbmay need to take this into account, for both preclinical and clinicalstudies. Indeed since glucan synergizes equally well with IgM antibody,the presence of natural IgM anti-tumor and anti-viral antibodies can bea confounding factor in interpreting in vivo tumor response, whether inpreclinical models or in clinical trials, unless the po intake of glucanin mouse chow is standardized. Most importantly, since many carbohydratetumor vaccines (e.g. GM2-KLH,³⁵ GD2-KLH, MUC-1,³⁶ andglobo-H-hexasaccharide³⁷) induce primarily specific IgM response, glucanmay enhance their anti-tumor effects. If this glucan effect can begeneralized to other antibody-mediated host defense mechanisms, its rolein infectious disease may also be intriguing. Serotherapy of certaindrug resistant bacteria,³⁸ or viral (e.g. CMV) and fungal (e.g.cryptococcus and candida ³⁹) infections using antibodies may be enhancedby concurrent intake of β-glucan. One can speculate if the function ofpre-existing protective antibodies, e.g. towards tetanus orstreptococcus, can be enhanced by oral β-glucan; indeed, if it canenhance the protective effects of common bacterial vaccines. Thesuccessful treatment of Alzheimer's disease using antibodies specificfor amyloid β-peptide in the mouse model is a provocative finding⁴⁴; itis likely that β-glucan may enhance the antibody effect. When oneconsider glucan-effect in the context of auto-immune disease, it is alsoplausible that tissue injury may be increased by oral glucan, leading toexacerbations of such diseases as rheumatoid arthritis. It is possiblein those auto-immune diseases in which auto-antibodies cause tissuedamage, clinical signs and symptoms may be modulated by oral intake ofglucan. In view of these potential beneficial and adverse effects ofbarley glucan on human diseases, a better understanding of their immuneeffects seems highly worthwhile.

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Purpose: In vitro β-glucan can enhance tumor cytotoxicity through iC3breceptors on leukocytes. We test if (1→3), (1→4)-β-D-glucan (β-glucan)can synergize with anti-GD2 monoclonal antibody (MoAb) 3F8 (mouse IgG3)in therapy of human neuroblastoma xenografts.

Experimental Design: Athymic nude mice with established neuroblastomaxenografts were treated with daily intraperitoneal or oral β-glucan, inthe presence/absence of intravneous MoAb twice a week, for 22-29 days.Serial tumor volumes and body weights were monitored.

Results: 3F8 plus β-glucan produced near-complete tumorregression/disease stabilization, while 3F8 or β-glucan alone did notsignificantly affect tumor growth. For NMB7 tumors, median survival of3F8 plus β-glucan group was 5.5 fold that of control groups (p<0.001),and for LAN-1 the survival difference was 2.6 fold. 47% of the mice withNMB7 and 18% with LAN-1 remained progression-free in contrast to <3% ofcontrols. Antitumor effect was seen at 340 ug glucan dose, i.v. or po,and in all human neuroblastoma cell lines tested. No toxicities werenoted in mice treated with either β-glucan alone or 3F8 plus β-glucan (4to 4000 ug per dose). In contrast to anti-GD2 MoAb 3G6 (IgM), 3F8F(ab′)₂ and MoAb 8H9 (IgG1) did not activate complement and had nosynergy with β-glucan. Anti-tumor effect of 3F8 plus oral β-glucanpersisted after anti-asialo-GM1 antibody treatment, as well as inNK-deficient host.

Conclusions: Oral 1,3-1,4-β-glucan synergized with anti-tumor IgG andIgM MoAb in vivo. Since β-glucan was well tolerated and inexpensive, itspotential value in cancer therapy deserves further investigation.

Introduction

Monoclonal antibodies (MoAb) selective for tumors have therapeuticpotential (1). The introduction of hybridoma technology by Kohler andMilstein in 1975 (2) and advances in molecular biologic techniques havegreatly expanded the potential of MoAb in human cancers. Evidence ofefficacy in clinical trials is increasingly evident: 17-1A in coloncancers (3), anti-CD20 in lymphoma (4, 5), anti-HER2 antibodies inbreast cancer (6, 7), and M195 against CD33 in acute leukemia (8) aregood examples. Our laboratory has developed the MoAb 3F8 which targetsthe ganglioside GD2 overexpressed on neuroblastoma. 3F8 has been shownto have high specificity and sensitivity in the radioimmunodetection ofminimal residual disease in patients with NB (9), and a significantclinical impact when used as adjuvant therapy (10).

The immune basis of clinical tumor response to MoAb is at leasttwo-fold, direct cytotoxicity and induced immunity. Antibody dependentcell-mediated cytotoxicity (ADCC) and complement mediated cytotoxicity(CMC) are responsible for the direct killing of tumor cells. On theother hand, through tumor opsonization (11) or idiotype network (12),tumor-specific immunity is induced. β-glucans are polymers of glucoseextractable from cereals, mushrooms, seaweed and yeasts (13). They are(1→3)-β-D-glucopyranosyl polymers with randomly dispersed singleβ-D-glucopyranosyl units attached by (1→6)-linkages, giving a comb-likestructure. The (1→3)-β backbone and the (1→6)-linked branches werethought to be important for their immune effects. Lentinan (fromLentinus edodes, Basidiomycete family) is a high molecular weight (MW)β-glucan with (1→6) branches off every three (1→3)-β-D-glucopyranosylresidues and it has been licensed in Japan for cancer treatment.Schizophyllan (from Schizophyllum commune, Basidiomycete family) andβ-glucan from Baker's yeast (Saccharomyces cerevisiae) have similarstructures. Laminarin (from seaweed), a small MW β-glucan, has (1→6)-βbranches occurring at every ten (1→3)-β-D glucopyranosyl units. On theother hand, β-glucan from barley, oat or wheat has mixed (1→3) and(1→4)-β-linkage in the backbone, but no (1→6)-β branches and aregenerally of high MW. Although barley (1→3), (1→4)-β-D-glucan has beenshown in vitro to bind to CR3 (14), activate ADCC mediated by naturalkiller cells (15-17), monocytes (18, 19), and neutrophils (17, 19), aswell as stimulating tumor necrosis factor (TNFα) production by monocytes(20), their in vivo immunomodulatory effects in cancer models have yetto be investigated.

We now report our findings that oral (1→3), (1→4)-β-D-glucan derivedfrom barley or oats can greatly enhance the activity of anti-tumormonoclonal antibodies in xenograft models. Because β-glucan is nontoxic,well tolerated and inexpensive, its role in cancer therapy deservescareful study.

Materials and Methods

Cell lines Human neuroblastoma cell lines LAN-1 were provided by Dr.Robert Seeger, Children's Hospital of Los Angeles, Los Angeles, Calif.,and NMB7 by Dr. Shuen-Kuei Liao (McMaster University, Ontario, Canada).Neuroblastoma cell lines SK-N-JD, SK-N-ER, and SK-N-MM were establishedfrom patients with metastatic disease treated at MemorialSloan-Kettering Cancer Center (MSKCC), New York, N.Y. Cell lines werecultured in 10% defined calf serum (Hyclone, Logan, Utah) in RPMI with 2mM L-glutamine, 100 U/ml of penicillin (Sigma, St. Louis, Mo.), 100ug/ml of streptomycin (Sigma), 5% CO₂ in a 37° C. humidified incubator.Normal human mononuclear cells were prepared from heparinized bonemarrow samples by centrifugation across a Ficoll density separationgradient.

Antibodies Monoclonal antibodies 3F8 (mouse IgG3), 3G6 (mouse IgM), and8H9 (mouse IgG1) reactive with neuroblastoma have been previouslydescribed (21, 22). They were produced as ascites and purified byaffinity chromatography: protein A (Pharmacia, Piscataway, N.J.) for 3F8(21), protein G (Pharmacia) for 8H9 (22), and C1q-sepharose (Pierce,Rockford, Ill.) for 3G6 (21, 23). These antibodies were >90% pure bySDS-PAGE. F(ab′)₂ fragments were prepared by pepsin digestion aspreviously reported (24). TIB114 (N.S.7), a hybridoma secreting an IgG3control antibody, was obtained from American Type Culture Collection(ATCC). Rabbit anti-asialo-GM1 antibody (Wako Pure Chemical Industries,Osaka, Japan) diluted to 1 mg/ml of protein was administered at 200 ulip on days 0, 1, 2, 7, 14, 21.

Glucan (1→3), (1→4)-β-D-glucan derived from barley and (1→4)-β-D-mannanwere purchased from Sigma. Sugar composition and linkage analysis wereperformed by the Complex Carbohydrate Research Center, University ofGeorgia, Athens, Ga., supported in part by the Department ofEnergy-funded Center for Plant and Microbial Complex Carbohydrates(DF-FG09-93ER-20097). Barley glucan was dissolved by boiling for 10minutes in normal saline.

Mice and treatment Athymic Balb/c mice were purchased from NCI,Frederick, Md., and CB-17 SCID-Beige mice from Taconic (Germantown,N.Y.). Tumor cells were planted (1-5×10⁶ cells) in 100 ul of Matrigel(BD BioSciences, Bedford, Mass.) subcutaneously. Following implantation,tumor sizes (maximum width and lengths) were measured. Tumor size wascalculated as product of the 2 perpendicular diameters. Treatmentstudies started in groups of 4-5 mice per cage when tumor diameterreached 0.7 to 0.8 cm, usually by 14-21 days of tumor implantation. Micereceived antibody treatment intravenously (generally 200 ug per dose byretroorbital injection) twice weekly and β-glucan by intragastric gavage(generally 400 ug per dose) every day for 3 weeks (22-days of β-glucanand 6-doses of antibody). Mice were weighed once a week and tumor sizemeasured twice a week. Mice were sacrificed when tumors reached sizesthat interfered with their well-being.

Assays for soluble cytokines Sera from mice were obtained 1 h, 4 h, 8 h,24 h, 48 h, and 72 h after oral β-glucan. They were assayed for solublecytokine IL-12 (p70) and TNFα, all reagents from Endogen (Woburn,Mass.). Briefly, 96-well microtiter plates were coated with eithermonoclonal anti-mouse IL12 at 5 ug/ml or monoclonal anti-TNFα at 0.8ug/ml overnight at ambient temperature. The mouse IL12 standard rangedfrom 1000 pg/ml in 1:3 serial dilutions and the TNFα standard rangedfrom 490 pg/ml in 1:2 serial dilution. Test samples (serum diluted 1:2)were added to the plates and incubated for 2 hours at ambienttemperature. The detecting antibody, biotinylated anti-mouse IL12monoclonal at 1:100 dilution for the IL12 assay, or biotinylatedanti-mouse TNFα monoclonal at (1:50) for the TNFα ELISA was added. Theplates were incubated at ambient temperature for one hour. After PBSwash, the secondary antibody, which was HRP-conjugated streptavidin at1:400 for IL12, and 1:200 for TNFα, was added to the plates for a 30 minincubation at ambient temperature. After another wash,tetramethylbenzidine was added as the substrate for the color reactionfor 30 min, and absorbance was read at 450 nm using an ELISA platereader. The limits of detection were 12 pg/ml for the mouse IL12 ELISA,and 10 pg/ml for the mouse TNFα ELISA.

Immunostaining for tumor vasculature LAN-1 xenografts were removed 1 h,4 h, 8 h, 16 h, 24 h, 48 h, 96 h and 216 h after treatment. Tumorvasculature was assayed by immunostaining with an anti-blood vesselantibody. Eight mm cryostat frozen tumor sections were fixed in acetoneand washed in PBS. Endogenous peroxidases were blocked in 0.3% H₂O₂ inPBS. Sections were incubated in 3% bovine serum albumin containing 0.25%gelatin for 60 minutes, after the avid-biotin blocking step. Incubationwith the biotinylated rat anti-murine PECAM IgG2a MoAb, MEC13.3 (1mg/ml) (BD PharMingen, San Diego, Calif.) was carried out at roomtemperature for 60 minutes followed by ABC complex (Vector Laboratories,Burlingame, Calif.). Color was developed with DAB peroxidase substratekit (Vector). A 10% hematoxylin counterstain for 4 minutes was used.

Statistical analysis Average tumor size over time between groups wascompared. The null hypothesis was no difference in size over time. Totest the hypothesis, the square of size differences summed over time wasused, which in effect compared the trajectories of the average tumorsizes between treatment groups.

${SS\_ DEV}\overset{k}{\underset{i = 1}{=}}{3\mspace{11mu}\left( {x_{i} - y_{i}} \right)^{2}}$where there were k time points and x_(i) and y_(i) were the averagetumor sizes at time i for each treatment group.Results

Synergy between barley β-glucan and anti-GD2 antibody 3F8 in eradicatinghuman neuroblastoma. 3F8 is a murine IgG3 monoclonal antibody thatactivates mouse and human complement, and mediates effective ADCCagainst human neuroblastoma cells in vitro. β-glucan when administeredorally at 400 ug per day had no appreciable effect on NMB7 tumor growthas did antibody 3F8 given i.v. alone. However, when β-glucan and 3F8were used in combination, tumor growth was near totally suppressed.In >47% of mice, tumors remained permanently suppressed followingtreatment. Similar observations were made with neuroblastoma cell linesderived from different sources: LAN-1 (FIG. 21 a), NMB7 (FIG. 21 b)SK-N-ER (FIG. 21 c), SK-N-MM and SK-N-JD (data not shown). β-glucan wasequally effective when administered orally or intraperitoneally. Incontrast, for the GD2-negative rhabdomyosarcoma, HTB82, 3F8 plusβ-glucan treatment was ineffective (data not shown). In addition, TIB114(IgG3 control) plus barley β-glucan, or 3F8 plus mannan had noanti-tumor (data not shown). When 3F8 dose was decreased from 200 ug to40 ug, the anti-tumor effect was lost (data not shown). There was nodetectable serum IL-12 or TNF-α release following oral β-glucanadministration (data not shown). There was no immunohistochemicallydetectable effect of β-glucan on tumor vessel formation (data notshown).

Dose response curve for ip β-glucan. When the dose of intraperitonealβ-glucan was decreased by 10-fold from 4000 ug, it was clear that 4 ugwas no longer effective in synergizing with MoAb 3F8 in suppressing NMB7growth. Interestingly, both ip and oral 1,3-1,4-β-glucan (at 400 ug perday) were effective (FIG. 22).

Oral β-glucan versus ip β-glucan. When oral β-glucan was studied in NMB7tumors (FIG. 23), similar dose response was found. While an oral dose of400 ug was curative for some tumors, breakthroughs were seen for lowerdose levels, with those receiving 4 ug escaping sooner those receiving40 ug. Using the LAN-1 tumor model, neither 4 nor 40 ug β-glucan wereeffective (data not shown). There was no significant body weight changein any of the treatment groups (after accounting for tumor weight),irrespective of β-glucan dose or co-administration with 3F8. At necropsyon day 22, there were no appreciable differences in the peripheral bloodcounts, cholesterol and blood chemistry between mice receiving differentβ-glucan doses. There was also no difference in the histologicappearances of organs in mice treated with β-glucan at any of the doselevels, when compared to control mice that received saline.

By the oral route, daily β-glucan schedule was necessary. A 5 day/weekpo β-glucan regimen was comparable to the daily regimen. In contrast, aonce a week or twice a week schedule of β-glucan had no anti-tumoreffect (data not shown).

Role of NK cells in β-glucan effect. Removal of NK cells by anti-AsialoGM1 antiserum eliminated a substantial amount, although not completelythe anti-tumor activity of β-glucan (FIG. 24). Moreover, in SCID-beigemice which lack NK cells, β-glucan was still effective in synergizingwith 3F8 (data not shown), suggesting that at least part of theanti-tumor activity was mediated by NK-independent cytotoxicity.

IgG3-F(ab′)₂ or IgG1 antibodies did not have anti-tumor activity. Therole of Fc in mediating the anti-tumor effect of β-glucan was apparentwhen Fc was removed by pepsin or when IgG1 isotype MoAb 8H9 was used(data not shown). Neither was able to activate complement or mediateefficient ADCC, and neither has significant anti-tumor effect whenadministered with 400 ug of oral β-glucan.

Synergy of β-Glucan with 3F8 in prolonging survival. Nude mice (n=22)with established neuroblastoma NMB7 xenografts (0.7-0.8 cm diametertumor at the beginning of treatment) were treated with 3F8 (200 ug twicea week iv) and 400 ug of β-glucan po daily for a total of 3 weeks.Control mice received either saline alone (n=10), 3F8 alone (n=8), orβ-glucan (n=16) alone. Median survival was 30 days in control groups and166 days in the treatment (3F8 plus β-glucan, n=22) group (p<0.001).Long-term survival was estimated at 47% in the treatment group and 3% inthe control group (saline alone, 3F8 alone, or β-glucan alone) (FIG.25). Similar experiments were carried out in nude mice bearingestablished LAN-1 xenografts (also 0.7-0.8 cm diameter tumor at thebeginning of treatment). Among control mice treated with either salinealone (n=31), 3F8 alone (n=16), or β-glucan (n=8) alone, tumor growthwas rapid. Median survival was 21 days in control groups (n=55) and 54days in the treatment (3F8 plus β-glucan) group (n=82) (p<0.001, FIG.26). Long-term survival was estimated at 18% in the treatment group and0% in the controls.

Discussion

Using the human xenograft models, we have made the followingobservations. β-Glucan derived from barley can synergize with monoclonalantibodies to suppress and/or eradicate tumors, while β-glucan orantibody alone has little anti-tumor effect. Anti-tumor responserequires antibodies that activate complement, and both mouse IgM andmouse IgG3 were effective. Oral administration of β-glucan is at leastequally (if not more) effective than the intraperitoneal route. It is adose-dependent phenomenon, where ≧400 ug per dose is required formaximal effect. Natural killer cells are not essential for this β-glucanphenomenon, although they contribute to the anti-tumor effect. NormalT-cells and B-cells are not required for the anti-tumor effect sinceimmune-deficient mouse strains demonstrate the β-glucan effect, in bothathymic and SCID-beige mice. Most importantly, oral β-glucan iswell-tolerated by all the mice tested so far, with no noticeable changein body weight, blood counts or organ histologies, even at doses as highas 4 mg per dose per day.

Our findings differ significantly from previous observations andpredictions on the use of β-glucans in cancer treatment. In the past itwas thought that the (1→3), (1→6)-β linkage was absolutely required forthe β-glucan anti-tumor effect (13). This structure contains(1→3)-β-D-glucopyranosyl units along which are randomly dispersed singleβ-D-glucopyranosyl units attached by (1→6)-β linkages, giving acomb-like structure (e.g. Lentinan, Schizophyllan, Laminarin, and glucanfrom Baker's yeast). In these models, it was believed that T-cells wereactivated and indeed required for the anti-tumor effect. In addition, itwas believed that small molecular weight β-glucan should be moreeffective than high molecular weight β-glucan and that the mosteffective administration should be intravenous or intraperitonealroutes. Indeed, Betafectin was derived from a genetically engineeredSaccharomyces cerevisiae which makes (1→3),(1→4)-β-D-glucans with weakerinterchain associations (25). It was manufactured for i.v. injection toimprove macrophage function in the hope of reducing infectiouscomplications and improving wound healing. Barley β-glucan is a linearpolymer with (1→3)-β and (1→4)-β linkages; however, it is not a comblike structure. We did not find any anti-tumor effect of barley β-glucanwhen given alone. However, when used in combinations with monoclonalantibodies, the synergistic effect was remarkable. Although β-glucanactivates granulocyte mediated ADCC in vitro (data not shown), theeffects of β-glucan may be indirect. It is not clear if the absorptionof β-glucan is necessary for its anti-tumor effect. The exact mechanismof how β-glucan enhances the anti-tumor effect of monoclonal antibodiesin vivo is unknown.

Monoclonal antibodies, either through Fc interaction or through CR3interaction with iC3b, target cytotoxicity to tumor cells, a processgreatly enhanced by β-glucan activation of effector cells. β-glucanactivates leukocytes by binding to CR3 or to β-glucan receptors (26).After antibodies deposit complement components on pathogens or cancercells, C3b is rapidly proteolyzed into iC3b fragment by serum factor I.These iC3b fragments then opsonize the pathogens or tumor cells for theiC3b-receptors (CR3, CD11b/CD18) on phagocytic cells and NK cells,stimulating phagocytosis and/or cytotoxic degranulation. Thus, antibodyand complement link innate and adaptive immunity by targeting antigensto different cells of the immune system, e.g. via CR3 and Fc forphagocytic cells, CR2 for B cells, and CR1, CR2, or CR3 for folliculardendritic cells (27). For neutrophils, CR3-dependent phagocytosisrequires ligation of two distinct binding sites, one for iC3b and asecond site for β-glucan. Without β-glucan, iC3b-opsonized target cellsare resistant to killing (17). Microbes possess polysaccharides that canactivate the lectin domain on CR3, leading to phagocytosis and cytotoxicdegranulation. In contrast, human cells (including tumors) lack theseCR3-binding polysaccharides, thus the inability of CR3 to mediatephagocytosis or extracellular cytotoxicity of tumor cells opsonized withiC3b. The lectin site of CR3 can also influence transmembrane signalingof endogenous neutrophil membrane GPI-anchored glycoproteins (e.g. CD14,CD16, and CD59). In a mouse mammary tumor model, where there arenaturally occurring IgM and IgG antibodies, injection of yeast solubleβ-glucan could suppress tumor growth, an effect lost in C3-deficient orCD11b (CR3)-deficient mice (28, 29). Since iC3b bound to a primaryprotein antigen can also enhance recognition and specific antibodysynthesis by antigen-specific B cells (30), the presence of β-glucanplus complement activation may enhance B-cell response to pathogens ortumor cells.

Another mechanism of action of β-glucan may relate to innate receptorsfor β-glucan, in a hard-wired information network on phagocytes andlymphoid cells; receptors that normally recognize death signals andmicrobial molecular patterns (31). These innate receptors are biosensorsfor invading pathogens widely distributed in vertebrate and invertebrateanimals (32), a nonclonal host defense pathway with structural andfunctional homologies in phylogenetic lineages that diverged over abillion years ago. Following β-glucan activation of leukocytes, killingis immediate, nonclonal, and obligatory, a process often referred to asinnate immunity. The consequence of this innate effector arm is theactivation of costimulatory molecules and induction of cytokines andchemokines that will enhance adaptive immunity to the tumor cells. (33,34). Thus, antibodies, complement, phagocytes, and “danger” receptorsform core elements of innate immunity while antigen-presenting cells, Tand B lymphocytes constitute essential players in acquired immunity.

Despite the availability of tumor-selective monoclonal antibodies andthe ample supply of phagocytes/natural killers, shrinkage of establishedtumors following antibody treatment alone, and the acquisition ofspecific immunity, is not common in both preclinical models and cancerpatients. The absence of a danger signal and the diminution ofcomplement action by complement resistance proteins on tumor cells mayexplain the inefficiency of antibody mediated clinical responses (35).Lipopolysaccharide and β-glucan, being cell wall components of bacteriaand fungus, respectively, are potent danger signals to the immunesystems in all life-forms, from Drosophila to man (36).

While LPS is too toxic for human use, β-glucan is relatively benign. Ifthis synergistic effect of β-glucan on antibodies is active in humans,our findings may have broad clinical implications. First the efficacy ofmonoclonal antibodies in cancer (e.g. Herceptin, Rituximab, Dacluzimab,anti-GD2 and anti-EGF-R MoAb) can be potentially enhanced (37).Nevertheless, even though toxicity from β-glucan is expected to beminimal, the enhanced efficacy of MoAb may also increase MoAb-mediatedtoxicity. For example, the side effects of Herceptin on cardiacfunction, or anti-GD2 MoAb on neuropathic pain may be increased. Second,since the amount and quality of barley and oat glucan in daily foodintake can vary, future interpretations of efficacy trials using MoAbmay need to take this into account, for both preclinical and clinicalstudies. Indeed since glucan synergizes equally well with IgM antibody,the presence of natural IgM anti-tumor and anti-viral antibodies can bea confounding factor in the interpretation of in vivo tumor response,whether in preclinical models or in clinical trials, unless the oralintake of glucan in mouse chow is standardized. Most importantly, sincemany carbohydrate tumor vaccines (e.g. GM2-KLH (38), GD2-KLH, andglobo-H-hexasaccharide (39)) primarily induce specific IgM response,glucan may enhance their anti-tumor effects. In view of these potentialbeneficial effects of barley glucan on cancer therapy, a betterunderstanding of their immune effects seems highly worthwhile.

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Background: β-glucan primes leukocyte CR3 for enhanced cytotoxicity andsynergizes with anti-tumor monoclonal antibodies (MoAb). We studied(1→3)-β-D-glucans in xenograft tumor models, and examined therelationship of its anti-tumor effect and physico-chemical properties.

Methods: Established subcutaneous human xenografts were treated withβ-glucan daily and MoAb twice weekly by intragastric injection for 29days. Control mice received either MoAb alone or β-glucan alone. Tumorsizes were monitored over time. β-glucans were studied by carbohydratelinkage analysis, and high performance size-exclusion chromatographywith multiple angle laser scattering detection.

Results: Orally administered β-D-glucan greatly enhanced the anti-tumoreffects of MoAb against established tumors in mice. This effectcorrelated with the molecular size of the (1→3),(1→4)-β-D-glucan. (1→3),(1→6)-β-D-glucans also synergized with MoAb, although the effect wasgenerally less. We observed this β-glucan effect irrespective of antigen(GD2, GD3, CD20, epidermal growth factor-receptor, HER-2), human tumortype (neuroblastoma, melanoma, lymphoma, epidermoid carcinoma and breastcarcinoma) or tumor sites (subcutaneous versus systemic).

Conclusion: Given the favorable efficacy and toxicity profile of oralβ-D-glucan, its role in cancer treatment as an enhancer of the effect ofMoAb therapy deserves careful study.

Introduction

Evidence of efficacy of monoclonal antibodies (MoAb) against humancancer in clinical trials is increasingly evident. However, induced oradministered antibodies to human tumors have not realized their fullesttherapeutic potential, even when they can activate complement-dependentcytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity(ADCC). The deposition of C3b and iC3b on tumor cells fails to stimulatephagocytosis or extracellular cytotoxicity by C3-receptor-bearingneutrophils, macrophages, and NK cells, even though these same effectorcells can efficiently kill C3b and iC3b opsonized microorganisms. Thereceptor for iC3b, CR3 (also called CD11b/CD18, Mac-1, orαMβ2-integrin), is found in monocytes/macrophages, NK cells, and immunecytotoxic T-lymphocytes. CR3 activation requires the engagement of twosites on its α-subunit (CD11b): the iC3b-binding site within theI-domain at the N-terminus and a lectin site at the C-terminus (1,2).β-glucans are specific for the lectin site. When coated with iC3b, yeastcells (with their β-glucan-containing cell wall), engage both iC3b andlectin binding sites on leukocytes, triggering phagocytosis andrespiratory burst (2,3). In contrast, tumor cells coated with iC3bcannot activate leukocytes because they lack the CR3-binding β-glucan(4-7). Soluble forms of β-glucans can bind to the lectin site (8,9) andprime both phagocytic and NK cells to kill iC3b coated tumor targets(4,9,10). In murine mammary tumor models in which iC3b was found,intravenous yeast β-glucan reduced tumor size by 70-95% (11). The lossof tumor response in the absence of complement-fixing IgM anti-tumorantibodies (SCID mice), or of C3 (C3 knockout mice), or of leukocyte CR3(CR3 knockout mice) highlighted the critical components of this iC3bstrategy (11).

Although (1→3)-β-D-glucans can be purified from yeast, seaweed andmushrooms, an inexpensive, convenient and safe source of pure(1→3),(1→4)-β-D-glucan is available from barley. Barley β-glucan hasbeen shown to bind to CR3 in vitro (9), to activate ADCC mediated by NKcells (4, 10, 12) monocyte (8,13), and neutrophils (8,14), as well as tostimulate TNF production by monocytes (15). However, its in vivoimmunomodulatory effects, especially when administered by the oralroute, have not been tested. In this study we report an unusually strongsynergism between anti-tumor antibodies and intragastric injection ofβ-glucan against a broad spectrum of human tumor xenografts. We alsoundertake a preliminary investigation of molecular size requirements forthis anti-tumor synergy with MoAb.

Methods

Cell lines The cell lines Daudi, RMPI 6666, HS455, SKMel-28 and A431were from American Type Culture Collection (ATCC), Rockville, Md. LAN-1was provided by Dr. R. Seeger, Children's Hospital of Los Angeles, LosAngeles, Calif.; NMB7 by Dr. S. K. Liao (McMaster University, Ontario,Canada); human breast carcinoma cell line BT474 was kindly provided byDr. David Solit of Memorial Sloan-Kettering Cancer Center (MSKCC), NewYork, N.Y.; SKNJD and SKNER were established at MSKCC. BT474 wascultured in Dulbecco's modified Eagle with Nutrient Mixture F12(DMEM/F-12) (Life Technologies Inc. GIBCO-BRL, Rockville, Md.) in a 1:1mixture fortified with 10% newborn calf serum (Hyclone, Logan, Utah),MEM non-essential amino acids (Gibco-BRL, Grand Island, N.Y.), 100 U/mlof penicillin (Sigma, St. Louis, Mo.), and 100 ug/ml of streptomycin(Sigma). All other cell lines were cultured in RPMI 1640 (LifeTechnologies Inc.) containing 10% defined calf serum (Hyclone) and 100U/ml of penicillin, 100 ug/ml of streptomycin and 2 mM L-glutamine(Sigma).

Antibodies MoAb 3F8 (mouse IgG3) and 3G6 (mouse IgM) reactive againstGD2 ganglioside expressed on neuroectodermal tumors, and MoAb 8H9 (mouseIgG1) reactive with a glycoprotein expressed on these same tumors havebeen previously described (16,17). They were purified to >90% purity byaffinity chromatography: protein A (Pharmacia, Piscataway, N.J.) for3F8, and protein G (Pharmacia) for 8H9. Anti-GD3 antibody (R24) (18) wasprovided by Dr. P. Chapman of MSKCC. Hybridomas producing theanti-epidermal growth factor receptor (EGF-R) antibodies 528 (IgG2a) and455 (IgG1) were obtained from ATCC (19). Rituximab (anti-CD20) andHerceptin (anti-HER2) were purchased from Genentech, San Francisco,Calif.

β-Glucan Barley, oat and lichenan β-D-glucans were purchased from Sigmaand Megazyme International Ireland Ltd., Wicklow, Ireland. Wheatβ-glucan was kindly provided by Dr. P. Wood of Agriculture and Agri-FoodCanada, West Guelph, Ontario. Betatrim (Quaker Oatrim, 5% β-glucan fromoat) was provided by Rhodia Food, Cranbury, N.J. Laminarin was purchasedfrom Sigma and from TCI America, Portland, Oreg. Lentinan (β-glucanextracted from the mushroom Lentinus edodes) was provided by the DrugSynthesis and Chemistry Branch, Developmental Therapeutics Program,Division of Cancer Treatment and Diagnosis, National Cancer Institute,Bethesda, Md. β-glucan was dissolved by boiling for 10 minutes in normalsaline; Lentinan was dissolved first in DMSO before diluting in water.Digestion with lichenase (endo-1,3:1,4-β-D-glucanase) from B. subtilis(Megazyme), was carried out in sodium phosphate buffer (20 mM, pH 6.5)at 40° C. for 10 minutes. Sugar composition and linkage analysis by gaschromatography-mass spectrometry following methylation was performed bythe Complex Carbohydrate Research Center, University of Georgia, Athens,Ga., supported in part by the Department of Energy-funded Center forPlant and Microbial Complex Carbohydrates (DF-FG09-93ER-20097) (20). Theaverage ratio of (1→3) to (1→4)-β-linkage in (1→3),(1→4)-β-D-glucansderived from barley, oat and wheat was around 3:7. For molecular sizeand shape estimations, β-glucan was analyzed by size-exclusionchromatography plus multiple-angle laser light scattering (MALLS) aspreviously described (21,22). Besides measuring molecular size by MALLS,the slope derived from root mean square radius versus molar-mass plotsgave an estimate of the molecular shape: a slope of 0.33 being the shapeof a sphere, 0.5 being random coils and 1.0 being rigid rods. High MWβ-glucans were found to be random coils in contrast to low MW specieswhich were more sphere-like.

Mice and treatment Athymic nu/nu mice were purchased from the NationalCancer Institute-Frederick Cancer Center (Bethesda, Md.) and ICR/SCIDfrom Taconic (White Plains, N.Y.) and maintained in ventilated cages.Experiments were carried out under Institutional Animal Care and UseCommittee (IACUC) approved protocols, and institutional guidelines forthe proper and humane use of animals in research were followed. Tumorcells were planted (1-5×10⁶ cells) in 100 μl of Matrigel (Sigma)subcutaneously. Tumor dimensions were measured two to three times a weekwith vernier calipers, and tumor size was calculated as the product ofthe two largest perpendicular diameters. For breast carcinoma xenograftstudies, 6-8 week old female nude mice (NCI) were initially implantedwith 0.72 mg 90-day release 17β-estradiol pellet (Innovative Research ofAmerica, Sarasota, Fla.) subcutaneously into the right flank.Twenty-four hours later, 10⁷ BT474 cells were implanted subcutaneouslyinto the left flank. All treatment studies started in groups of 4-5 micewhen tumor diameters reached 0.7 to 0.8 cm. Mice received antibodytreatment (40-200 ug per day) i.v. (by retroorbital injection) twiceweekly and oral β-glucan (400 ug per day) by intragastric injectionevery day for a total 4 weeks. Mice were weighed once a week andsacrificed according to IACUC guidelines. In the SCID mouse systemichuman lymphoma (Daudi) model, 5 million cells were administered i.v.,and treatment started 10 days later.

Statistical analysis Because measurement times varied betweenexperiments, and mice in control groups frequently were sacrificed [asrequired by IACUC for rapidly enlarging tumors] before the end of eachexperiment, tumor growth was calculated by fitting a regression slopefor each individual mouse to log transformed values of tumor size.Slopes were compared between groups using linear regression withmonoclonal antibody treatment, β-glucan treatment and combinationtreatment as covariates. In the study of melanoma tumor growth, β-glucanwas given at two different doses. Thus, dose was added as a covariatefor analysis. In the study of epidermoid tumor growth, monoclonalantibody was given at three different doses and antibody dose was addedas a covariate. Trends for slope by molecular weight were tested bylinear regression of slope scores. Survival analysis was conducted byCox regression using the indicator variables: monoclonal antibodytreatment, β-glucan treatment, and combination treatment; in thesurvival study of lymphoma, Rituximab was given at two different dosesand so dose of antibody was added as a covariate for analysis. Allanalysis were conducted using STATA (Stata Corporation, College Station,Tex.).

Results

Synergy between MoAb and barley β-glucan. We chose the mouse modelbecause of its relative inefficiency in CDC and ADCC (23), and MoAbalone were typically ineffective against established tumors. Oraladministration of β-glucan (average MW 210 kD) from barley alone at 400ug qd×29 days or antibody 3F8 i.v. alone had no appreciable effect onneuroblastoma LAN-1 tumor growth. The tumor growth rates for theβ-glucan alone, 3F8 alone, and saline controls were virtually identical.In contrast, when we combined oral β-glucan with i.v. 3F8, significantlyless tumor growth was observed in the 3F8 antibody alone group, 0.7% vs5.4% increase in tumor size per day, respectively. In the regressionmodel, only combination treatment significantly reduced tumor growth(4.9% per day, 95% CI 2.4%, 7.4%, p=0.001). Nude mice (n=22) withestablished NMB7 xenografts were treated with 3F8 (200 ug twice a weekiv) and 400 ug of β-glucan po daily for a total of 4 weeks. Control micereceived either saline alone, 3F8 alone, or β-glucan alone. Mediansurvival was 30 days in control groups and 166 days in the treatment(3F8 plus β-glucan, n=22, FIG. 27). In the Cox model, combinationβ-glucan/antibody treatment was the only variable significantlyassociated with improved survival (hazard ratio treatment: 0.07, 95% CI0.02, 0.27; p<0.0001). Ten (45%) mice in the combination group survivedlong term with a median follow-up of 248 days. Only one mouse in any ofthe control groups (<5%) remained alive during the experiment. Thisanti-tumor effect was evident against a panel of GD2-positiveneuroblastoma lines: NMB7, SK-N-JD, and SK-N-ER. Barley β-glucan waseffective when the route of administration was either intragastric orintraperitoneal. In contrast, if the tumor was antigen-negative (e.g.GD2-negative rhabdomyosarcoma HTB82), 3F8 plus β-glucan treatment wasineffective. When the dose of oral β-glucan was decreased by 10-foldsfrom 4000 ug to 400 ug, 40 ug and 4 ug, the tumor growth rate were4.3±2.2%, 3.8±0.9%, 8.1±0.8%, and 9.5±0.9%, respectively. These datasuggest an optimal dose somewhere between 400 ug and 4000 ug. Theanimals did not suffer weight loss, or histopathologic changes in themajor organs at necropsy in the treatment groups, irrespective ofβ-glucan dose. When the glucan was heated at 95° C. up to 3 hours, itsin vivo effects remained intact. However, following digestion withendo-(1→3), (1→4)-β-D-glucanase, all its in vivo effect was destroyed.In addition, the anti-tumor effect of the antibodies was lost when theFc of the antibody was removed by pepsin, or when an IgG1 isotype (Moab8H9) was used.

Molecular size of (1→3),(1→4)-β-D-glucan and anti-tumor synergy withMoAb. Barley β-glucans of increasing molecular sizes were tested at anoral dose of 40 ug or 400 ug. Anti-tumor effect improved with increasingmolecular size, with a 2.2% decrease in tumor growth rate per day foreach increase of 100 kD in molecular weight; 95% confidence interval3.0%, 1.4%; p<0.00001 (Table 1). However, since the shape of theβ-glucan in aqueous solution correlated with average MW by MALLSanalysis, potency could be a function of molecular shape rather thanmolecular size.

Source of β-D-glucan and anti-tumor synergy with MoAb. A (1→3),(1→4)-β-D-glucan of average MW of 210 kD derived from barley was chosenas our standard. Using the neuroblastoma xenograft model, equivalent ugdoses of β-glucans derived from various plant sources were compared intheir anti-tumor activity when administered by intragastric injectionplus intravenous MoAb 3F8 (Table 1). As expected, since the chemicalcomposition (1→3),(1→4)-β-D-glucan derived from barely, oat and wheatwere similar, comparable levels of synergy with MoAb was found, and highMW also appeared to be more effective. When glucans with(1→6)-β-linkages were tested, high MW species (e.g. Lentinan 1,500 kD)was not as effective compared to the standard. On the other hand, low MW(1→3),(1→6) preparations (e.g. Laminarin 5 kD), though not as effectiveas standard, was more potent than low MW (1→3),(1→4)-β-D-glucan.β-glucan effect in a wide spectrum of MoAbs and human tumor models.Using the standard β-glucan from barley (210 kD), a series of MoAb werescreened against a panel of human tumor xenografts in various mousestrains. The combination of oral β-glucan with complement activatingMoAb suppressed tumor growth significantly in contrast to MoAb orβ-glucan alone. This was shown for anti-GD3 MoAb (R24) against melanoma(FIG. 28A), anti-EGF-R (528) MoAb against epidermoid carcinoma A431(FIG. 28B), and anti-HER2 (Herceptin) against human breast carcinomaBT474 xenografts in nude mice (FIG. 28C). Again, MoAb 455, an IgG1anti-EGF-R was ineffective against epidermoid carcinoma in contrast tothe IgG2a 528 (FIG. 28B). In metastatic lymphoma model, Daudi cellsinjected i.v. established widespread tumors in brain, spinal cord,kidneys and ovaries of SCID mice (data not shown). In the Cox model,only combination treatment and dose of Rituximab were associated withsurvival. Median survival was 59 days in animals receiving eitherRituximab alone, β-glucan alone or no treatment. Median survival in thegroup treated with Rituximab plus β-glucan was 97 days (hazard ratio0.09; 95% CI 0.03, 0.27; p<0.001).

Discussion

We have shown that β-glucans greatly enhanced the anti-tumor effects ofMoAb against established tumors in mice. We observed this effectirrespective of route of β-glucan administration (intragastric orintraperitoneal), antigen (GD2, GD3, CD20, EGFR, HER2), human tumor type(NB, melanoma, epidermoid carcinoma, lymphoma, breast cancer), mousestrain (athymic nu/nu, severe combined immune deficiency mice), or tumorsite (subcutaneous versus systemic). β-glucan was heat-stable, itsanti-tumor effect was dose- and schedule-dependent, requiringantibody-Fc, but not cytophilicity of the antibody. Neither antibody norβ-glucan alone was effective. We detected no toxicities even at β-glucandoses of 4000 ug/day for 4 weeks. This synergy of (1→3),(1→4)-β-D-glucanwith MoAb increased with β-glucan MW.

β-glucans have been tested for tumor therapy in mice for nearly 40 years(24,25). Several forms of mushroom-derived β-glucans are used clinicallyin Japan to treat cancer, including polysaccharide Kureha (PSK, fromCoriolus versicolor), Lentinan and Schizophyllan. In randomized trialsin Japan, PSK and Schizophyllan have moderately improved survival ratesin some cancer trials (26-30), and less encouraging in others (31,32).While β-glucans are not used by western oncologists, β-glucan containingbotanical medicines such as Ling-Zhi, maitake and green barley arewidely used by cancer patients in the US as alternative/complementarycancer therapies, often with poor clinical validation or qualitycontrol.

Given the biology of iC3b targeted cytotoxicity, β-glucan should haveclinical potential. However, limitations with existing β-glucanstrategies are several fold. They are generally expensive andinconvenient to administer: e.g. Lentinan and Schizophyllan are giveni.v. daily over long periods of time. Besides being insoluble, theycontain proteins and non-β-glucan carbohydrates, which confoundmechanistic studies and complicate the manufacturing and controlprocess. Because of protein contaminants they are potentiallyallergenic. The spontaneous cross-linking of CR3 by β-glucan of high MWcan cause neutrophil degranulation and cytokine release frommacrophages, resulting in undesirable clinical toxicities. For low MWβ-glucan, besides their low affinity for CR3, they have rapid renalclearance. Without anti-tumor antibodies to activate human complement,β-glucan is largely ineffective. Here we addressed these limitations by(1) using pure (1→3),(1→4)-β-D-glucan from barley, (2) administeringβ-glucan orally instead of intravenously, and (3) coadministration oftumor-specific antibodies to ensure complement activation.

Previous studies have demonstrated that oral β-glucans activate splenicand peritoneal macrophages for tumor-cytotoxicity. In a study of¹⁴C-labeled oral β-glucan, sequestration in the liver was observed (33),suggesting that oral β-glucan entered the blood and behavedpharmacokinetically similar to intravenously administered low MWβ-glucan (34-36). These studies also suggested that processing by thegastrointestinal tract produced β-glucan with high activity for CR3.Besides this model of intravasation of processed barley β-glucan,leukocytes could also be activated directly in the gut before homing tothe tumor. It is of interest that unpurified β-glucan (Betratrim) haslow biologic activity in our model. Despite the abundance of β-glucan(3% of the dry weight) in grains, its bioavailability from cereals islimited since high MW β-glucans requires high temperature (>60° C.)extraction and final gelling. It is therefore not surprising thathigh-fiber (13.5 g/day) wheat-bran supplement did not have anti-tumoreffects in recent human trials (37).

Our findings using (1→3), (1→4)-β-D-glucans from barley were unexpected.In previous studies, the comb-like branch structure of (1→3),(1→6)-β-linkage (e.g. lentinan, schizophyllan, laminarin, and β-glucanfrom Baker's yeast) was deemed requisite for its anti-tumor effect (38).In those models, however, T-cells were essential. In our studies, (1→3),(1→4)-β-glucan could reproducibly enhance the anti-tumor effect of MoAbin immunodeficient mice, clearly demonstrating that neither T nor Bcells were needed. Although the absolute proof of complement and CR3requirement would have to await experiments with knock-out mice,preliminary evidence from studying MoAb isotypes and subclasses didsuggest that complement activation was required. Since most cancersexpress mCRP (CD46, CD55, CD59) on their cell surface (39-46),complement mediated tumor lysis is typically inefficient. Nevertheless,despite these inhibitory proteins, iC3b has been detected on tumor cellsisolated from fresh human breast tumors, and enough levels could bedeposited by MoAb (e.g. Herceptin) in vitro to opsonize tumor cells forphagocytes and NK cells in vitro (47). It is possible that sublethallevels of complement activation deposited enough iC3b to optimize tumorkilling, a strategy that deserves clinical investigation.

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TABLE 1 Neuroblastoma growth rate (%/day) when treated with i.v. 3F8 andoral β-glucan derived from various plant sources. 400 ug**** 40 ug**** %tumor growth Glucans Description MW (kD) relative to standard(1→3),(1→4)-β-D-glucan* Standard barley glucan 210 100 100 Antibodyalone Control — 287 — Saline control — 307 — Barley MW standard 337 7959 MW standard 237 100 100 MW standard 178 117 520 MW standard 131 163481 MW standard 48 180 516 BBG111 266 60 242 BBG126 90 146 — Oat BBG128262 88 — BBG127 201 104 — Wheat BBG117 138 — 190 Betatrim Unpurified —325 — Lichenan BBG113 132 189 — (163),(166)-β-D-glucan Laminarin**BBG108 5 177 Laminarin*** BBG109 32 326 Lentinan BBG114 1491 123*(163)β-linkage was ~30% for all the (163),(164)-β-D-glucans**(163)β-linkage was 92% ***163)β-linkage was 53% ****Either 400 or 40ug of (16 3)-β-D-glucans was administered orally qd × 29 days plusintravenous MoAb 3F8 twice a week (M, Th) × 8 doses in groups of 4-5mice each. Tumor size was measured periodically over the entiretreatment period. Tumor growth curve and potency were calculated asdetailed in Materials and Methods.Fourth Series of ExperimentsPhase I Study of Oral β-glucan and Intravenous Anti-GD2 MonoclonalAntibody 3F8 Among Patients with Metastatic Neuroblastoma

The main findings are as follows:

The clinical response data for the first eight patient shows thatβ-glucan has been extremely well-tolerated. There have been nodose-limiting toxicities. There is a somewhat bitter flavor, but thiscan be easily masked by chewing gum.

The in vitro correlates suggests that biologic activity of β-glucan withantibody-dependent cell cytotoxicity has been enhanced by treatment.

The patients are all children or adolescents with relapsed or refractorystage 4 neuroblastoma metastatic to bone, marrow or distant lymph nodes,some with large soft tissue masses. Even though these patients have beentreated with only 1-2 cycles of antibody+glucan at the lowest dose, wehave seen 6/8 patients demonstrating radiographic or histologicresponse.

Results and Discussion

Toxicities:

Glucan Dose level: #patients DLT I 6 0 II 2 0In Vitro Biologic Correlates:

Some initial data are available from the in vitro cytotoxicity studies.Though the number of patients is very small, some interesting trends areapparent. In particular, granulocyte mediated antibody-dependentcellular cytotoxicity (ADCC) directed at complement sensitized tumortargets approximately doubled from baseline values after just 3 days ofβ-glucan therapy (p=0.01 by Wilcoxon signed-rank test). Although thereare insufficient data for inferential comparisons, we can compare theseresults with those from a previous trial at MSKCC in which GM-CSF wasused concurrently with 3F8. Increases in ADCC averaged ˜40% in thesepatients, suggesting that β-glucan enhanced ADCC more consistently.

Clinical tumor response after cycle #1 Tumor Dose level Patient Bonemarrow MIBG CT markers I 1 NE Improved Improved NE 2 SD SD PD PD 3 CRImproved PD CR 4 SD Improved SD Improved 5 PR Improved NE CR 6 SDImproved Improved SD II 1 SD SD SD SD 2 — — PD — CR = completeremission, NE = not evaluable (no evaluable disease), PD = progressivedisease, PR = partial remission, from diffuse involvement to a singlefocus of tumor on biopsy, SD = stable disease

These clinical responses are highly promising given that they aredemonstrated after 1-2 cycles of antibody+glucan in a Phase I trial.(FIG. 29A) shows extensive osseous metastasis in the femora, fibulae,pelvis, rib, left scapula, right clavicle, humeri, skull and spine.Heart, liver, stomach and colon uptakes are physiologic. (FIG. 29B)shows a significant improvement of the patient 5 two months after asingle cycle of therapy. This is uncommon in our 13 years of experienceusing 3F8 in patients with refractory or relapsed metastatic stage 4neuroblastoma.

Fifth Series of Experiements

Rituximab activates complement-mediated and antibody-dependentcell-mediated cytotoxicities, and is effective against B-cell lymphomas.β-glucans are naturally occurring glucose polymers that bind to thelectin domain of CR3, a receptor widely expressed among leukocytes,priming it for binding to iC3b activated by antibodies. Barley-derived(1→3),(1→4)-β-D-glucan (BG), when administered orally (400 μg per day×29days), strongly synergized with subtherapeutic doses of intravenousrituximab (200 μg twice/week×8 doses) in the therapy of CD20-positivehuman lymphomas. Growth of established subcutaneous non-Hodgkin'slymphoma (NHL) (Daudi and EBV-derived B-NHL) or Hodgkin's disease (Hs445or RPMI6666) xenografted in SCID mice was significantly suppressed, whencompared to mice treated with rituximab or BG alone. Survival of micewith disseminated lymphoma (Daudi and Hs445) was significantlyincreased. There was no weight loss or clinical toxicity in treatedanimals. This therapeutic efficacy and lack of toxicity of BG plusrituximab supports further investigation into its clinical utility.

Introduction

The chimeric anti-CD20 antibody rituximab is being evaluated in anincreasing number of disorders. After clinical efficacy was initiallydemonstrated against relapsed and refractory follicular/low gradenon-Hodgkin's lymphoma¹, responses to rituximab have been reported inother malignant and non-malignant B-cell disorders². Several mechanismsof action have been proposed including activation of apoptoticpathways³, elaboration of cytokines⁴, and elicitation of hostcomplement-dependent cytotoxicity (CDC) and antibody-dependentcell-mediated cytotoxicity (ADCC)⁵. Although many patients with B-celldisorders respond to rituximab, remissions are often transient⁶. Morethan 50% of lymphomas recurrent after rituximab treatment failed torespond the second time⁷. Mechanisms of resistance to rituximab are asyet unclear, and may include paucity or loss of target antigen⁸,pharmacokinetic variations among individual patients, FcR polymorphism⁹,resistance to complement activity¹⁰, or inherent gene expression of thelymphoma¹¹.

β-glucans are complex polymers of glucose with affinity for the lectinsite of the CR3 receptor on leucocytes¹². With bound β-glucan, CR3(CD11b) is primed to engage iC3b fragments deposited on cells bycomplement-activating antibodies. This receptor mediates the diapedesisof leukocytes through the endothelium and stimulates phagocytosis,degranulation and tumor cytotoxicity. Many fungi present β-glucan orβ-glucan-like CR3 binding ligands on their cell surface. Hence, wheniC3b deposition occurs, both CD11b and lectin sites become engaged, andphagocytosis and respiratory burst is triggered¹³. In contrast, tumorcells lack such molecules, and even when coated with iC3b do notgenerally activate CR3 and cannot activate leucocytes. Soluble forms ofβ-glucan bind to lectin sites and prime both phagocytic and NK cells tokill iC3b-coated tumor targets¹⁴.

(1→3), (1→4)-D-β-glucan (BG), a soluble, barley-derived β-glucan hasadvantages over previously studied (1→3), (1→6)-β-glucans, particularlyefficacy when administered orally and a good safety profile¹⁵. In vivosynergism between BG and the complement-fixing antibody 3F8 againsthuman neuroblastoma xenografts^(15,16) was recently demonstrated. Thesynergism between BG and rituximab against lymphoma is now reported.

Study Design

Cell Lines:

Human Burkitt's lymphoma cell line, Daudi, and Hodgkin's disease (HD)cell lines Hs445 and RPMI 6666 were purchased from American Type CultureCollection (Rockville, Md.). Human EBV-BLCL were established usingpreviously described methods¹⁷.

Mice:

Fox Chase ICR SCID mice (Taconic, White Plains, N.Y.) were maintainedunder institutionally approved guidelines and protocols.

Tumor Models:

Subcutaneous tumors were established by injecting 5×10⁶ cells suspendedin 0.1 ml of Matrigel (Becton-Dickinson, Franklin Lakes, N.J.) into miceflanks. Tumor dimensions were measured two to three times a week andtumor size was calculated as the product of the two largest diameters.Mice were sacrificed when maximum tumor dimension exceeded 20 mm. Adisseminated tumor model was established in SCID mice as previouslydescribed¹⁸. Briefly, 5×10⁶ Daudi or Hs445 cells in 100 μl normal salinewere injected intravenously into SCID mice. Tumors grew systemically andmice became paralyzed when tumor cells infiltrated the spinal cord,resulting in hind-leg paralysis. Mice were sacrificed at onset ofparalysis or when animals lost 10% of their body weight.

Treatment Regimens:

For mice with subcutaneous tumors, therapy was initiated after tumorswere established (7-8 mm diameter). For the disseminated tumor model,therapy was initiated ten days after injection of tumor cells. Groups ofat least five mice per treatment regimen received either rituximab, BG,neither or both. 200 μg rituximab (Genentech, San Francisco, Calif.) wasinjected intravenously twice weekly for a total of eight injections and400 μg BG (Sigma, St. Louis, Mo.) administered orally via intragastricgavage daily for 29 days. Animals were weighed weekly and observedclinically at least once daily.

Statistical Analysis:

Tumor growth was calculated by fitting a regression slope for eachindividual mouse to log transformed values of tumor size. Slopes werecompared between groups using t-tests using a previously describedmethod for censored observations¹⁹. Survival in mice with disseminateddisease was compared using Kaplan-Meier analysis and proportion ofdeaths was compared by Fisher's exact χ² test. Analyses were conductedusing STATA 7 (Stata Corporation, College Station, Tex.).

Results and Discussion

In all subcutaneous xenograft models, significant reduction in tumorgrowth was noted in mice treated with a combination of rituximab and BG.Mice treated with rituximab alone showed a modest reduction in tumorgrowth, while those treated with BG alone or left untreated had unabatedtumor growth (FIGS. 30A, 30B, 30C). All tumors except for those treatedwith combination therapy grew beyond 20 mm size and mice had to besacrificed. Mice on combination treatment had persistent tumorsuppression even after treatment was stopped. In a multivariable linearmodel of tumor growth rate, using dummy variables for treatment, theinteraction between BG and rituximab was positive and significant,demonstrating synergism.

For disseminated xenografts, there was a significant difference insurvival between the combination and control groups for both NHL and HDmodels (p<0.005, by log-rank) (FIG. 31A and 31B). 5/38 mice and 2/8 micewith disseminated Daudi and Hs445 tumors respectively treated withcombination BG and rituximab were surviving >12 months after therapy wasdiscontinued suggesting complete eradication of disease. In contrast,0/29 and 0/8 mice receiving rituximab alone in respective groupssurvived (15% vs 0% survival; χ²=0.01). There was no significant weightloss or other clinically apparent adverse effects. That BG is absorbedcan be inferred from the fact that it could be detected intracellularlywithin fixed and permeabilized peripheral blood leucocytes byimmunofluorescence (data not shown).

In these studies, synergism between BG and rituximab was highlysignificant irrespective of the type of CD20-positive lymphoma. Improvedresponses in Daudi xenografts as compared to Hs445 may be attributableto higher CD20 expression in the former (Mean geometric fluorescencechannel for Daudi 241 compared to 184 for Hs445). When tumors thatprogressed were examined for CD20 expression by immunofluorescencestudies of single cell suspensions or indirect immunohistochemistry offrozen sections, no significant difference was noted between groupstreated with rituximab, BG alone or rituximab+BG (data not shown),indicating that treatment with rituximab+BG was not associated with lossof CD20.

Synergism between other complement-activating monoclonal antibodies andBG^(15,16) were previously demonstrated. The current data extend thisobservation to rituximab. CDC is considered an important mechanism forrituximab cytotoxicity. Rodent complement is not inhibited efficientlyby human complement regulatory proteins (mCRP). Therefore CDC can be aneffective anti-tumor mechanism in xenograft models. However in ourstudy, at sub-therapeutic doses of antibody, rituximab-mediated ADCC andCDC were not sufficient to effect tumor cell killing. Since BG has nodirect effect on ADCC²⁰, this synergy is most likely a result ofiC3b-mediated tumor cytotoxicity. Lymphoma cells express mCRP includingCD46, CD55, and CD59^(10,21). However, iC3b-mediated cytotoxicity isunaffected by the presence of CD59 which affects only MAC-mediatedcomplement cytotoxicity²². Furthermore, in human breast carcinomatumors, deposition of iC3b has been demonstrated despite the presence ofmCRP²³ indicating that unlike their inhibitory effect on MAC, effect oniC3b-mediated tumor cytotoxicity is not absolute.

If this synergistic effect can be safely reproduced in humans,iC3b-mediated cytotoxicity may be a potential strategy to overcomerituximab resistance in patients with B-cell malignancies. Since neitherT nor B cells are required for this synergistic effect, BG may have apotential role even in immunocompromised lymphoma patients. Furthermore,in patients with autoimmune disorders, B-cell depletion may be enhancedwith this non-toxic oral therapy. Conversely, β-glucans can enhancerelease of cytokines such as TNF-α and IL-6²⁴, and because the acutetoxicities of rituximab are also related to cytokine release secondaryto complement activation²⁵, there is a potential of increased toxicitywhen BG and rituxirnab are used in combination. Carefully designed phaseI studies are necessary in order to define the safety and efficacy indeveloping BG as an adjunct to rituximab therapy in the treatment ofB-cell disorders and in antibody-based therapies of other cancers.

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1. A method for enhancing the anti-tumor or anti-cancer effect of anantibody administered to a subject comprising the steps of, orallyadministering to said subject a glucan derived from barley in an amounteffective to enhance the anti-tumor effect of a complement-activatingantibody, wherein the antibody binds to a cancer cell expressing anantigen selected from the group consisting of CD20, HER2, EGFR, GD2, andGD3, wherein the glucan is a β-glucan having a backbone consisting ofβ-(1,3) and β-1,4) glycosidic linkages.
 2. The method of claim 1,wherein the antibody is a monoclonal antibody.
 3. The method of claim 1,wherein the antibody is capable of activating antibody dependentcell-mediated cytotoxicity.
 4. The method of claim 1 wherein thecomplement activating antibody and the glucan are administered to thesubject concurrently or sequentially.
 5. The method of claim 1, whereinthe cancer cell is selected from the group consisting of a neuroblastomacell, a melanoma cell, a non-Hodgkin's lymphoma cell, a Hodgkin'slymphoma cell, an Epstein-Barr related lymphoma cell, an epidermoidcancer cell, and a breast cancer cell.
 6. The method of claim 1, whereinthe amount of glucan administered is about >=25 mg/kg/day, five days aweek for a total of 2-4 weeks.
 7. The method of claim 1, wherein thesubject is a mammal.
 8. The method of claim 7, wherein the subject is ahuman.
 9. A method for enhancing the anti-tumor or anti-cancer effectsof an antibody administered to a subject comprising the steps of, orallyadministering to said subject a glucan derived from barley in an amounteffective to enhance the anti-tumor effect of a complement-activatingantibody, wherein the antibody binds to a cancer cell selected from thegroup consisting of neuroblastoma, melanoma, breast cancer, lymphoma andepidermoid cancers, wherein the glucan is a β-glucan having a backboneconsisting of β-(1,3) and β-1,4) glycosidic linkages.
 10. The method ofclaim 9, wherein the antibody is a monoclonal antibody.
 11. The methodof claim 9, wherein the antibody is capable of activating antibodydependent cell-mediated cytotoxicity.
 12. The method of claim 9 whereinthe complement activating antibody and the glucan are administered tothe subject concurrently or sequentially.
 13. The method of claim 9,wherein the neuroblastoma cell expresses the antigen GD2.
 14. The methodof claim 9, wherein the melanoma cell expresses the antigen GD3.
 15. Themethod of claim 9, wherein the lymphoma cell expresses the antigen CD20.16. The method of claim 15, wherein lymphoma cell is selected from thegroup consisting of non-Hodgkin's lymphoma, Epstein-Barr relatedlymphoma, and Hodgkin's lymphoma.
 17. The method of claim 9, wherein theamount of glucan administered is about >=25 mg/kg/day, five days a weekfor a total of 2-4 weeks.
 18. The method of claim 9, wherein the subjectis a mammal.
 19. The method of claim 18, wherein the subject is a human.